Wei Seong Toh

Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells

Human embryonic stem cells (hESCs) have the potential to offer a virtually unlimited source of chondrogenic cells for use in cartilage repair and regeneration. We have recently shown that expandable chondrogenic cells can be derived from hESCs under selective growth factor-responsive conditions. In this study, we explore the potential of these hESC-derived chondrogenic cells to produce an extracellular matrix (ECM)-enriched cartilaginous tissue construct when cultured in hyaluronic acid (HA)-based hydrogel, and further investigated the long-term reparative ability of the resulting hESC-derived chondrogenic cell-engineered cartilage (HCCEC) in an osteochondral defect model. We hypothesized that HCCEC can provide a functional template capable of undergoing orderly remodeling during the repair of critical-sized osteochondral defects (1.5 mm in diameter, 1 mm depth into the subchondral bone) in a rat model. In the process of repair, we observed an orderly spatial-temporal remodeling of HCCEC over 12 weeks into osteochondral tissue, with characteristic architectural features including a hyaline-like neocartilage layer with good surface regularity and complete integration with the adjacent host cartilage and a regenerated subchondral bone. By 12 weeks, the HCCEC-regenerated osteochondral tissue resembled closely that of age-matched unoperated native control, while only fibrous tissue filled in the control defects which left empty or treated with hydrogel alone. Here we demonstrate that transplanted hESC-derived chondrogenic cells maintain long-term viability with no evidence of tumorigenicity, providing a safe, highly-efficient and practical strategy of applying hESCs for cartilage tissue engineering.

Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment

An injectable and biodegradable hydrogel system comprising hyaluronic acid-tyramine (HA-Tyr) conjugates can safely undergo covalent cross-linking in vivo by the addition of small amounts of peroxidase and hydrogen peroxide (H(2)O(2)), with the independent tuning of the gelation rate and degree of cross-linking. Such hydrogel networks with tunable mechanical and degradation properties may provide the additional level of control needed to enhance chondrogenesis and overall cartilage tissue formation in vitro and in vivo. In this study, HA-Tyr hydrogels were explored as biomimetic matrices for caprine mesenchymal stem cells (MSCs) in cartilage tissue engineering. The compressive modulus, equilibrium swelling and degradation rate could be controlled by varying the concentration of H(2)O(2) as the oxidant in the oxidative coupling reaction. Cellular condensation reflected by the increase in effective number density of rounded cells in lacunae was greater in softer hydrogel matrices with lower cross-linking that displayed enhanced scaffold contracture. Conversely, within higher cross-linked matrices, cells adopted a more elongated morphology, with a reduced degree of cellular condensation. Furthermore, the degree of hydrogel cross-linking also modulated matrix biosynthesis and cartilage tissue histogenesis. Lower cross-linked matrix enhanced chondrogenesis with increases in the percentage of cells with chondrocytic morphology; biosynthetic rates of glycosaminoglycan and type II collagen; and hyaline cartilage tissue formation. With increasing cross-linking degree and matrix stiffness, a shift in MSC differentiation toward fibrous phenotypes with the formation of fibrocartilage and fibrous tissues was observed. These findings suggest that the tunable three-dimensional microenvironment of the HA-Tyr hydrogels modulates cellular condensation during chondrogenesis and has a dramatic impact on spatial organization of cells, matrix biosynthesis, and overall cartilage tissue histogenesis.

Effects of Culture Conditions and Bone Morphogenetic Protein 2 on Extent of Chondrogenesis from Human Embryonic Stem Cells

The study of human embryonic stem cells (hESCs) can provide invaluable insights into the development of numerous human cell and tissue types in vitro. In this study, we addressed the potential of hESCs to undergo chondrogenesis and demonstrated the potential of hESC‐derived embryoid bodies (EBs) to undergo a well‐defined full‐span chondrogenesis from chondrogenic induction to hypertrophic maturation. We compared chondrogenic differentiation of hESCs through EB direct‐plating outgrowth system and EB‐derived high‐density micromass systems under defined serumfree chondrogenic conditions and demonstrated that cell‐tocell contact and bone morphogenetic protein 2 (BMP2) treatment enhanced chondrocyte differentiation, resulting in the formation of cartilaginous matrix rich in collagens and proteoglycans. Provision of a high‐density three‐dimensional (3D) microenvironment at the beginning of differentiation is critical in driving chondrogenesis because increasing EB seeding numbers in the EB‐outgrowth system was unable to enhance chondrogenesis. Temporal order of chondrogenic differentiation and hypertrophic maturation indicated by the gene expression profiles of Col 1, Col 2, and Col 10, and the deposition of extracellular matrix (ECM) proteins, proteoglycans, and collagen II and X demonstrated that the in vivo progression of chondrocyte maturation is recapitulated in the hESC‐derived EB model system established in this study. Furthermore, we also showed that BMP2 can influence EB differentiation to multiple cell fates, including that of extraembryonic endodermal and mesenchymal lineages in the EB‐outgrowth system, but was more committed to driving the chondrogenic cell fate in the EB micromass system. Overall, our findings provide a potential 3D model system using hESCs to delineate gene function in lineage commitment and restriction of chondrogenesis during embryonic cartilage development.

Combined effects of TGFβ1 and BMP2 in serum-free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation

This study investigated the effects of TGFβ1, BMP2 or a combination of both on the chondrogenic differentiation of mesenchymal stem cells (MSCs) in a serum-free micromass culture system in vitro. Putative MSCs harvested from the iliac crest of 4–5 month old New Zealand White Rabbits were expanded and cultured in three-dimensional high density micromass aggregate cultures containing TGFβ1, BMP2 or a combination of both, in the absence of serum. After 14–20 days of culture, chondrogenic differentiation of the MSCs was assayed by toluidine blue staining, immunohistochemistry and semi-quantitative RT-RCR of type I collagen (CI) and type II collagen (CII). Quantitative measurements of cell proliferation and sulfated glycosaminoglycan (s-GAG) were also carried out to assess the growth rate and matrix deposition of the cultured aggregates. Both immunohistochemical staining and semi-quantitative RT-PCR showed that the combination of BMP2 and TGFβ1 resulted in a marked enhancement of collagen II synthesis, with minimal collagen I expression, which would suggest hyaline-like cartilage formation. Additionally, BMP2 and TGFβ1 had a synergistic effect on matrix proteoglycan deposition, as assessed by metachromatic toluidine blue staining. This is consistent with the quantitative measurement of glycosaminoglycans, whereby a significant increase in GAG/DNA was noted in the co-treatment group. Hence, it can be concluded that the combination of BMP2 and TGFβ1 has a synergistic effect on the differentiation of MSC into hyaline-like cartilage tissue.

Advances in hydrogel delivery systems for tissue regeneration.

Hydrogels are natural or synthetic polymer networks that have high water-absorbing capacity and closely mimic native extracellular matrices. As hydrogel-based cell delivery systems are being increasingly employed in regenerative medicine, several advances have been made in the hydrogel chemistry and modification for enhanced control of cell fate and functions, and modulation of cell and tissue responses against oxidative stress and inflammation in the tissue environment. This review aims to provide the state-of-the-art overview of the recent advances in field, discusses new perspectives and challenges in the regeneration of specific tissues, and highlights some of the limitations of current systems for possible future advancements.

Potential of Human Embryonic Stem Cells in Cartilage Tissue Engineering and Regenerative Medicine

The current surgical intervention of using autologous chondrocyte implantation (ACI) for cartilage repair is associated with several problems such as donor site morbidity, de-differentiation upon expansion and fibrocartilage repair following transplantation. This has led to exploration of the use of stem cells as a model for chondrogenic differentiation as well as a potential source of chondrogenic cells for cartilage tissue engineering and repair. Embryonic stem cells (ESCs) are advantageous, due to their unlimited self-renewal and pluripotency, thus representing an immortal cell source that could potentially provide an unlimited supply of chondrogenic cells for both cell and tissue-based therapies and replacements. This review aims to present an overview of emerging trends of using ESCs in cartilage tissue engineering and regenerative medicine. In particular, we will be focusing on ESCs as a promising cell source for cartilage regeneration, the various strategies and approaches employed in chondrogenic differentiation and tissue engineering, the associated outcomes from animal studies, and the challenges that need to be overcome before clinical application is possible.

Differentiation and enrichment of expandable chondrogenic cells from human embryonic stem cells in vitro

Human embryonic stem cells (hESCs) are considered as useful tools for pre‐clinical studies in regenerative medicine. Although previous reports have shown direct chondrogenic differentiation of mouse and hESCs, low yield and cellular heterogenicity of the resulting cell population impairs the generation of sufficient numbers of differentiated cells for further testing and applications. Based on our previously established high‐density micromass model system to study hESC chondrogenesis, we evaluated the effects of transforming growth factor (TGF)‐β1 and bone morphogenetic protein‐2 on early stages of chondrogenic differentiation and commitment by hESCs. Significant chondrogenic induction of hESCs, as determined by quantitative measurements of cartilage‐related gene expression and matrix protein synthesis, was achieved in the presence of TGF‐β1. By means of selective growth factor combination (TGF‐β1, FGF‐2 and platelet‐derived growth factor‐bb) and plating on extracellular matrix substratum, we report here the reproducible isolation of a highly expandable, homogenous and unipotent chondrogenic cell population, TC1, from chondrogenically committed hESCs. Like primary chondrocytes, TC1 rapidly dedifferentiates upon isolation and monolayer expansion but retains the chondrogenic differentiation potential and responds to TGF‐β1 for cartilaginous tissue formation both in vitro and in vivo. In addition, TC1 displays a somatic cell cycle kinetics, a normal karyotype and does not produce teratoma in vivo. Thus, TC1 may provide a potential source of chondrogenic cells for drug testing, gene therapy and cell‐based therapy.

Biomaterial-Mediated Delivery of Microenvironmental Cues for Repair and Regeneration of Articular Cartilage

Articular cartilage injuries are one of the most challenging problems in musculoskeletal medicine due to the poor intrinsic regenerative capacity of this tissue. The lack of efficient treatment modalities motivates research into tissue engineering: combining cells, biomaterials mimicking extracellular matrix (scaffolds) and microenvironmental signaling cues. The aim of this review is to focus on the use of biomaterials as delivery systems for microenvironmental cues in relation to their applications for treatment of cartilage defects. The latest advances in cartilage tissue engineering and regeneration are critically reviewed to demonstrate an outline of challenges toward biomaterial-based approaches of cartilage regeneration.

Advances in Mesenchymal Stem Cell-based Strategies for Cartilage Repair and Regeneration

Significant research efforts have been undertaken in the last decade in the development of stem cell-based therapies for cartilage repair. Among the various stem cell sources, mesenchymal stem cells (MSCs) demonstrate great promise and clinical efficacy in cartilage regeneration. With a deeper understanding of stem cell biology, new therapeutics and new bioengineering approaches have emerged and showed potential for further developments. Of note, there has been a paradigm shift in applying MSCs for tissue regeneration from the use of stem cells for transplantation to the use of stem cell-derived matrix and secretome components as therapeutic tools and agents for cartilage regeneration. In this review, we will discuss the emerging role of MSCs in cartilage regeneration and the most recent advances in development of stem cell-based therapeutics for cartilage regeneration.

The effect of injectable gelatin-hydroxyphenylpropionic acid hydrogel matrices on the proliferation, migration, differentiation and oxidative stress resistance of adult neural stem cells

Transplanted or endogenous neural stem cells often lack appropriate matrix in cavitary lesions in the central nervous system. In this study, gelatin-hydroxyphenylpropionic acid (Gtn-HPA), which could be enzymatically crosslinked with independent tuning of crosslinking degree and gelation rate, was explored as an injectable hydrogel for adult neural stem cells (aNSCs). The storage modulus of Gtn-HPA could be tuned (449-1717 Pa) to approximate adult brain tissue. Gtn-HPA was cytocompatible with aNSCs (yielding high viability >93%) and promoted aNSC adhesion. Gtn-HPA demonstrated a crosslinking-based approach for preconditioning aNSCs and increased the resistance of aNSCs to oxidative stress, improving their viability from 8-15% to 84% when challenged with 500 μM H(2)O(2). In addition, Gtn-HPA was able to modulate proliferation and migration of aNSCs in relation to the crosslinking degree. Finally, Gtn-HPA exhibited bias for neuronal cells. In mixed differentiation conditions, Gtn-HPA increased the proportion of aNSCs expressing neuronal marker β-tubulin III to a greater extent than that for astrocytic marker glial fibrillary acidic protein, indicating an enhancement in differentiation towards neuronal lineage. Between neuronal and astrocytic differentiation conditions, Gtn-HPA also selected for higher survival in the former. Overall, Gtn-HPA hydrogels are promising injectable matrices for supporting and influencing aNSCs in ways that may be beneficial for brain tissue regeneration after injuries.