Bogyu Choi

Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering

Injectable cartilaginous constructs that can form gels in tissue defects have many advantages in tissue engineering applications. In this study we created an injectable hydrogel consisting of methacrylated glycol chitosan (MeGC) and hyaluronic acid (HA) by photocrosslinking with a riboflavin photoinitiator under visible light. A minimum irradiation time of 40s was required to produce stable gels for cell encapsulation with 87-90% encapsulated chondrocyte viability. Although increasing the irradiation time from 40 to 600 s significantly enhanced the compressive modulus of the hydrogels up to 11 or 17 kPa for MeGC or MeGC/HA, respectively, these conditions reduced the encapsulated cell viability to 60-65%. The majority of chondrocytes encapsulated in MeGC hydrogels after 300 s irradiation maintained a rounded shape with a high cell viability of ~80-87% over a 21 day culture period. The incorporation of HA in MeGC hydrogels increased the proliferation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. These findings demonstrate that MeGC/HA composite hydrogels have the potential for cartilage repair.

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.

Visible light crosslinkable chitosan hydrogels for tissue engineering.

In situ gelling constructs, which form a hydrogel at the site of injection, offer the advantage of delivering cells and growth factors to the complex structure of the defect area for tissue engineering. In the present study, visible light crosslinkable hydrogel systems were presented using methacrylated glycol chitosan (MeGC) and three blue light initiators: camphorquinone (CQ), fluorescein (FR) and riboflavin (RF). A minimal irradiation time of 120 s was required to produce MeGC gels able to encapsulate cells with CQ or FR. Although prolonged irradiation up to 600 s improved the mechanical strength of CQ- or FR-initiated gels (compressive modulus 2.8 or 4.4 kPa, respectively), these conditions drastically reduced encapsulated chondrocyte viability to 5% and 25% for CQ and FR, respectively. Stable gels with 80-90% cell viability could be constructed using radiofrequency (RF) with only 40s irradiation time. Increasing irradiation time up to 300s significantly improved the compressive modulus of the RF-initiated MeGC gels up to 8.5 kPa without reducing cell viability. The swelling ratio and degradation rate were smaller at higher irradiation time. RF-photoinitiated hydrogels supported proliferation of encapsulated chondrocytes and extracellular matrix deposition. The feasibility of this photoinitiating system as in situ gelling hydrogels was further demonstrated in osteochondral and chondral defect models for potential cartilage tissue engineering. The MeGC hydrogels using RF offer a promising photoinitiating system in tissue engineering applications.

Customized biomimetic scaffolds created by indirect three-dimensional printing for tissue engineering.

Three-dimensional printing (3DP) is a rapid prototyping technique that can create complex 3D structures by inkjet printing of a liquid binder onto powder biomaterials for tissue engineering scaffolds. Direct fabrication of scaffolds from 3DP, however, imposes a limitation on material choices by manufacturing processes. In this study, we report an indirect 3DP approach wherein a positive replica of desired shapes was printed using gelatin particles, and the final scaffold was directly produced from the printed mold. To create patient-specific scaffolds that match precisely to a patients external contours, we integrated our indirect 3DP technique with imaging technologies and successfully created custom scaffolds mimicking human mandibular condyle using polycaprolactone and chitosan for potential osteochondral tissue engineering. To test the ability of the technique to precisely control the internal morphology of the scaffolds, we created orthogonal interconnected channels within the scaffolds using computer-aided-design models. Because very few biomaterials are truly osteoinductive, we modified inert 3D printed materials with bioactive apatite coating. The feasibility of these scaffolds to support cell growth was investigated using bone marrow stromal cells (BMSC). The BMSCs showed good viability in the scaffolds, and the apatite coating further enhanced cellular spreading and proliferation. This technique may be valuable for complex scaffold fabrication.

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.

Anionic carbohydrate-containing chitosan scaffolds for bone regeneration

Scaffolds derived from naturally occurring polysaccharides have attracted significant interest in bone tissue engineering due to their excellent biocompatibility and hydrophilic nature favorable for cell attachment. In this study, we developed composite chitosan (CH) scaffolds containing anionic carbohydrate, such as chondroitin 4-sulfate (CS) or alginate (AG), with biomimetic apatite layer on their surfaces, and investigate their capacity to deliver progenitor cells (bone marrow stromal cells, BMSC) and model proteins with net-positive (histone) and net-negative charge (bovine serum albumin, BSA). The incorporation of CS or AG in CH scaffolds increased compressive modulus of the scaffolds and enhanced apatite formation. Initial burst release of histone was significantly higher than that of BSA from CH scaffold, while the addition of CS or AG in the scaffolds significantly reduced the initial burst release of histone, indicating strong electrostatic interaction between histone and negatively charged CS or AG. The apatite layer created on scaffold surfaces significantly reduced the initial burst release of both BSA and histone. Furthermore, apatite-coated scaffolds enhanced spreading, proliferation, and osteogenic differentiation of BMSC seeded on the scaffolds compared to non-coated scaffolds as assessed by live/dead and alamarBlue assays, scanning electron microscopy (SEM), alkaline phosphatase (ALP) activity, and Picrosirius red staining. This study suggests that apatite-coated CH/CS composite scaffolds have the potential as a promising osteogenic system for bone tissue engineering applications.

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.

Novel role for cyclophilin A in regulation of chondrogenic commitment and endochondral ossification.

ABSTRACT Recent studies showed that cyclophilin A (CypA) promotes NF-κB/p65 nuclear translocation, resulting in enhanced NF-κB activity and altered expression of its target genes, such as the Sox9 transcriptional factor, which plays a critical role in chondrogenic differentiation and endochondral ossification. In this report, we unveil the role of CypA in signal-induced chondrogenic differentiation and endochondral ossification. Expression levels of the chondrogenic differentiation markers and transcriptional regulators Sox9 and Runx2 were all significantly lower in CypA knockdown chondrogenic cells than in wild-type cells, indicating that CypA plays a functional role in chondrogenic differentiation. In vitro differentiation studies using micromass cultures of mouse limb bud cells further supported the conclusion that CypA is needed for chondrogenic differentiation. Newborn CypA-deficient pups double stained with alcian blue and alizarin red exhibited generalized, pronounced skeletal defects, while high-resolution micro-computed tomography (microCT) analyses of the femurs and lumbar vertebrae revealed delayed or incomplete endochondral ossification. Comparative histology and immunohistochemistry (IHC) analyses further verified the effects of CypA deficiency on chondrogenic differentiation. Our results provide evidence for the important contribution of CypA as a pertinent component acting through NF-κB–Sox9 in regulation of chondrogenesis signaling. These findings are important to better understand signal-induced chondrogenesis of chondrogenic progenitors in physiological and pathophysiological contexts.