Cleo Choong

PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages.

We compared the transcriptomes of marrow-derived mesenchymal stem cells (MSCs) with differentiated adipocytes, osteocytes, and chondrocytes derived from these MSCs. Using global gene-expression profiling arrays to detect RNA transcripts, we have identified markers that are specific for MSCs and their differentiated progeny. Further, we have also identified pathways that MSCs use to differentiate into adipogenic, chondrogenic, and osteogenic lineages. We identified activin-mediated transforming growth factor (TGF)-beta signaling, platelet-derived growth factor (PDGF) signaling and fibroblast growth factor (FGF) signaling as the key pathways involved in MSC differentiation. The differentiation of MSCs into these lineages is affected when these pathways are perturbed by inhibitors of cell surface receptor function. Since growth and differentiation are tightly linked processes, we also examined the importance of these 3 pathways in MSC growth. These 3 pathways were necessary and sufficient for MSC growth. Inhibiting any of these pathways slowed MSC growth, whereas a combination of TGF-beta, PDGF, and beta-FGF was sufficient to grow MSCs in a serum-free medium up to 5 passages. Thus, this study illustrates it is possible to predict signaling pathways active in cellular differentiation and growth using microarray data and experimentally verify these predictions.

Pleiotrophin Enhances Clonal Growth and Long‐Term Expansion of Human Embryonic Stem Cells

To identify additional growth factors for optimizing propagation of human embryonic stem cells (hESCs), we mined publicly available data sets for the transcriptomes of murine and human ESCs and feeder cells, thereby generating a list of growth factors and complementary receptors. We identified the major pathways previously reported to be important, as well as several new ones. One pathway is the Pleiotrophin (PTN)‐Pleiotrophin receptor (PTPRZ1) axis. Murine fibroblasts secrete Ptn, whereas hESCs expressed PTPRZ1, which is downregulated upon differentiation. Depletion of PTPRZ1 resulted in decreased colony formation and lower recovery of hESCs. Supplementation of chemically defined medium for feeder‐free propagation of hESCs with PTN allowed higher recovery of hESCs without loss of pluripotency. PTN‐PTPRZ1 functions here predominantly via an antiapoptotic effect mediated in part by the activation of Akt. These findings reveal the underlying importance of PTN in hESC survival and its usefulness in the clonal manipulation and large‐scale propagation of hESCs.

PVDF film tethered with RGD-click-poly(glycidyl methacrylate) brushes by combination of direct surface-initiated ATRP and click chemistry for improved cytocompatibility

To enhance the cytocompatibility of polyvinylidene fluoride (PVDF) films, arginine–glycine–aspartic acid (RGD) peptide-click-poly(glycidyl methacrylate) (PGMA) polymer brushes were grafted onto the PVDF surface by the combination of surface-initiated atom transfer radical polymerization (ATRP) and click reaction. The direct initiation of the secondary fluorine atoms of PVDF backbone allowed for grafting of the PGMA brushes containing reactive epoxy groups. Subsequent introduction of the azide groups onto the side chain of PGMA brushes was achieved by the ring-open reaction of the epoxy groups with sodium azide. The cell adhesive RGD peptide was finally conjugated onto the PGMA brushes via alkyne-azide click reaction. Kinetic studies revealed that the PGMA chain growth from the PVDF surface was consistent with a “controlled” process, and that the amount of immobilized RGD peptide on the PGMA brushes increased with the concentration of the pendant azide groups. The specificity of cellular interactions of adipose tissue-derived stem cells (ASCs) on the functionalized PVDF films was investigated. Results demonstrated that cell adhesion and proliferation of ASCs were significantly improved on the RGD-immobilized PVDF substrates, and this improvement was positively correlated with the surface concentration of covalently-bonded RGD peptide. With the inherent superior chemical and mechanical properties of PVDF films and the biocompatible nature of cell-adhesive peptides, surface functionalized PVDF films are potentially useful for biomedical and tissue engineering applications.

Surface modification of polycaprolactone substrates using collagen-conjugated poly(methacrylic acid) brushes for the regulation of cell proliferation and endothelialisation

The incorporation and presentation of cell recognition ligands on the surfaces of biodegradable blood-vessel implants to promote endothelialisation is considered to be a promising approach to prevent platelet aggregation and hence thrombogenesis. In this study, cell-adhesive collagen was covalently immobilised onto polycaprolactone (PCL) substrates via surface-initiated atom transfer radical polymerization (ATRP) to improve cell–material interactions. Functional polymer brushes of poly(methacrylic acid) (P(MAA)) containing dense and reactive carboxyl groups (–COOH) were formed on the PCL substrates in a controllable manner. The amount of collagen, which was conjugated to the pendant carboxyl groups via carbodiimide chemistry, increased with the concentration of –COOH groups on the grafted P(MAA) brushes. The affinity and growth of endothelial cells (ECs) were found to be significantly improved on the collagen-immobilised PCL substrates, and this improvement is positively correlated with the amount of covalently conjugated collagen. Thus, surface-initiated ATRP provides an alternative methodology for the surface functionalisation of biodegradable polyester scaffolds to enable the formation of a confluent layer of ECs. An optimally endothelialised material surface will play a major role in the minimisation of thrombogenicity and inflammation, and hence can be potentially used for vascular graft applications.

Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Anti-cAngptl4 Ab-Conjugated N-TiO2/NaYF4:Yb,Tm Nanocomposite for Near Infrared-Triggered Drug Release and Enhanced Targeted Cancer Cell Ablation

Nanomedicine: NIR-active N-TiO(2) /NaYF(4) :Yb,Tm nanocomposites (NCs) were synthesized for the first time and its potential applications in drug release and targeted cancer cell ablation are explored. Upon 980 nm laser irradiation, the anti-cAngptl4 Ab-conjugated N-TiO(2) /NaYF(4) :Yb,Tm NCs shows a significant increase in apoptotic A-5RT3 cells when compared with that of the unconjugated NCs. The mechanisms for NIR-induced photocatalysis, drug release and targeted cancer cell killing are proposed.

Immobilization of Gelatin onto Poly(Glycidyl Methacrylate)-Grafted Polycaprolactone Substrates for Improved Cell–Material Interactions

To enhance the cytocompatibility of polycaprolactone (PCL), cell-adhesive gelatin is covalently immobilized onto the PCL film surface via two surface-modified approaches: a conventional chemical immobilization process and a surface-initiated atom transfer radical polymerization (ATRP) process. Kinetics studies reveal that the polymer chain growth from the PCL film using the ATRP process is formed in a controlled manner, and that the amount of immobilized gelatin increases with an increasing concentration of epoxide groups on the grafted P(GMA) brushes. In vitro cell adhesion and proliferation studies demonstrate that cell affinity and growth are significantly improved by the immobilization of gelatin on PCL film surfaces, and that this improvement is positively correlated to the amount of covalently immobilized gelatin. With the versatility of the ATRP process and tunable grafting efficacy of gelatin, this study offers a suitable methodology for the functionalization of biodegradable polyesters scaffolds to improve cell–material interactions.

Enhancing antibacterial activity of surface-grafted chitosan with immobilized lysozyme on bioinspired stainless steel substrates.

Bacterial infections have been widely recognized as a major cause of the failure of medical implants and devices. One promising strategy to reduce the incidence of infections is to impart the material surfaces with bactericidal function for inhibiting bacterial adhesion and biofilm formation. In this study, stainless steel (SS) surface was first activated by a biomimetic dopamine anchor to provide active amino groups, followed by covalently immobilizing chitosan (CS) with glutaraldehyde (GA) as a bifunctional linker. Hen egg white lysozyme, a natural defensive enzyme, was finally conjugated to the grafted chitosan to enhance biocidal functionality. The antibacterial assay results demonstrated substantial enhancement in bactericidal efficiency against Staphylococcus aureus (S. aureus) on the lysozyme-immobilized SS substrates under the neutral pH conditions as compared to the chitosan-grafted SS substrates. With the inherent advantages of robust anchoring ability of dopamine and specific functionality of lysozyme, the metallic substrates can be readily tailored with antibacterial property to combat biomaterial-centered infection for potential biomedical applications.

Preparation of a soft and interconnected macroporous hydroxypropyl cellulose methacrylate scaffold for adipose tissue engineering

This study describes the preparation and characterization of a biodegradable 3D hydrogel constructed from hydroxypropyl cellulose (HPC), modified with bifunctional methacrylic anhydride (MA) to form hydroxypropyl cellulose methacrylate (HPC-MA), for adipose tissue engineering applications. The hydrogels were prepared from three different concentrations (10 wt%, 15 wt% and 20 wt%) of HPC-MA with 0.35 degree of substitution. HPC-MA hydrogel scaffolds with open biphasic features were prepared by exploiting the thermal responsive phase behavior of HPC and temperature mediated phase separation of HPC-MA. The resulting scaffolds exhibited pore sizes ranging from 30 to 300 μm and an interconnected porosity of ∼90%. The swelling ratio (SR) and storage modulus of HPC-MA scaffolds were in the range of 12.94 to 35.83 and 0.75 to 4.28 kPa, respectively. The swelling ratio and storage modulus suggested that the scaffold exhibits high water retention, allowing medium exchange during cell culturing and that it is suitable for adipose tissue regeneration. The HPC-MA scaffolds were found to be biocompatible to human adipose-derived stem cells (ASCs). ASCs were successfully differentiated into the adipocytes inside the scaffolds, and therefore demonstrated the potential application of these HPC-MA scaffolds for adipose tissue engineering.

Multifunctional P(PEGMA)–REDV conjugated titanium surfaces for improved endothelial cell selectivity and hemocompatibility

Pre-vascularization of scaffolds using endothelial cells (ECs) and preservation of hemocompatibility are effective strategies to improve the long-term viability of tissue engineered constructs. The current work reports a multifunctional titanium (Ti) surface for simultaneous enhancement of EC selectivity while preserving hemocompatibility. This is achieved by REDV conjugation on surface-grafted PEGMA polymer brushes via surface-initiated atom transfer radical polymerization (ATRP) on a dopamine (DOPA)-modified Ti surface. Our results showed that the proliferation and attachment of human umbilical vein endothelial cells (HUVECs) were substantially improved by P(PEGMA)-REDV conjugation compared to pristine Ti surfaces, whilst no significant effects were observed for the mesenchymal stem cells (MSCs), thus confirming the selectivity of REDV for ECs. Platelets adhesion assay further revealed that the immobilization of PEGMA polymer brushes led to the amelioration of surface hemocompatibility, and this enhancement was not negated by the conjugation of REDV. The current multifunctional Ti-surface can potentially be useful in tissue engineered constructs for bone and dental applications as it allows for early and selective EC attachment and improved hemocompatibility whilst at the same time supporting MSC proliferation and growth.