Ting Ting Lau

Stromal cell-derived factor-1 (SDF-1): homing factor for engineered regenerative medicine

Introduction: Stromal cell-derived factor-1α (SDF-1) is a chemokine that plays a major role in cell trafficking and homing of CD34+ stem cells. Studies employing SDF-1/CXCR4 have demonstrated its therapeutic potential in tissue engineering. During injury, cells from the injured organ highly express SDF-1, which causes an elevation of localized SDF-1 levels. This leads to recruitment and retention of circulating CD34+ progenitor cells at the injury site via chemotactic attraction toward a gradient of SDF-1. The general approaches for SDF-1 introduction in tissue engineering are direct protein incorporation into scaffolds and transplantation of SDF-1-overexpressing cells and both methods are successful in improving the regeneration of the damaged tissue/organ. Areas covered: The mechanisms of SDF-1-mediated homing via CXCR4 receptor and the success of SDF-1-based medical applications in mesenchymal stem cell (MSC) homing as well as areas such as therapeutic angiogenesis, wound healing and neuronal and liver regeneration. Expert opinion: Current SDF-1 delivery designs and platforms hold much room for improvement. Regardless of the different techniques of SDF-1 introduction, they have proved to be effective in recruitment of various stem/progenitor cells. The pursuit of SDF-1-related regenerative medicine has already begun. It is thus conceivable that its usage in the clinical setting will be a reality in the near future.

Cytocompatibility study of a natural biomaterial crosslinker—Genipin with therapeutic model cells

Genipin has been widely used as a natural crosslinker to substitute chemical crosslinkers such as glutaraldehyde to crosslink various biomaterials like gelatin, collagen, and chitosan. However, there are contradicting views on the cytotoxicity and safety of genipin in tissue engineering. Therefore in this study, we aimed to evaluate the toxicity of genipin on skeletal tissues cells-osteoblasts and chondrocytes as they are also representatives of typical anchorage-dependent cells (ADCs) and nontypical ADCs. Results suggest that genipin toxicity is dose dependent and acute but not time dependent on both osteoblasts and chondrocytes. In particular, chondrocytes exhibit substantial alterations in the gene expression when exposed to Maximum nontoxic concentration (MaxNC) of genipin but there were no significant changes in the genes tested in osteoblasts. Since osteoblasts are typical ADCs, cellular focal adhesion assessment was carried out with F-actin being more contracted and unorganized when exposed to minimum toxic concentration (MinTC) of genipin. The mechanisms involved in cell deaths in both cell types are believed to be similar and hence using osteoblast as the model, cells were stained positive for Annexin-V and Reactive oxygen species (ROS) level were elevated at MinTC of genipin. Collectively, genipin induced cell apoptosis via ROS production, and apparently, gene expressions could also be altered at MaxNC. For this reason, we recommend the dose of genipin to be controlled within 0.5 mM.

A temperature-cured dissolvable gelatin microsphere-based cell carrier for chondrocyte delivery in a hydrogel scaffolding system☆

In this study, a novel therapeutic cell delivery methodology in the form of hydrogel encapsulating cell-laden microspheres was developed and investigated. As a model cell for cartilage tissue engineering, chondrocytes were successfully encapsulated in gelatin-based microspheres (mostly of diameter 50-100 μm, centred at 75-100 μm) with high cell viability during the formation of microspheres via a water-in-oil single emulsion process under a low temperature without any chemical treatment. These cell-laden microspheres were then encapsulated in alginate-based hydrogel constructs. By elevating the temperature to 37°C, the cell-laden microspheres were completely dissolved within 2 days, resulting in the same number of same-sized spherical cavities in hydrogel bulk, along with which the encapsulated cells were released from the microspheres and suspended inside the cavities to be cultivated for further development. In this cell delivery system, the microspheres played a dual role as both removable cell vehicles and porogens for creation of the intra-hydrogel cavities, in which the delivered cells were provided with both free living spaces and a better permeable environment. This temperature-cured dissolvable gelatin microsphere-based cell carrier (tDGMC) associating with cell-laden hydrogel scaffold was attempted and evaluated through WST-1, quantitative polymerase chain reaction, biochemical assays and various immunohistochemistry and histology stains. The results indicate that tDGMC technology can facilitate the delivery of chondrocytes, as a non-anchorage-dependent therapeutic cell, with significantly greater efficiency.

Inducing ossification in an engineered 3D scaffold-free living cartilage template

Large and complex bone defects or voids cannot rely on natural bone healing process for recovery. They require natural or engineered grafts to facilitate osteo-progenitor cell recruitment and development. In this study, we have employed an in vitro macro-sized 3D cell-based platform for investigation and application of osteogenesis. The model is based on a porous construct made of engineered living cartilaginous tissue named living hyaline cartilaginous graft (LhCG). It is scaffold-free and is solely made up of living chondrocytes and their extra cellular matrix (ECM). To evaluate the efficiency of LhCG as a viable platform for bone formation, osteoblast and human mesenchymal stem cell (hMSC) were seeded respectively into LhCG constructs, establishing a co-culture system consisting of osteo-progenitors and chondrocytes. The results showed that LhCG could support both osteoblast and hMSC maturation and differentiation to the osteogenic lineage respectively. Successful osteogenesis is also observed after subcutaneous implantation in nude mice model suggesting that bone formation could be achieved both in vitro and in vivo. Additionally, with exposure to osteogenic medium, LhCG construct without any further cell seeding expressed similar levels of osteogenic phenotype markers as the ones with hMSC seeded on. It suggests the existence of an osteoprogenitor sub-population residing within LhCG chondrocytes. Hence, it is demonstrated that LhCG, as a cartilage template, could serve as a dynamic platform to support osteogenesis and its intrinsic phenotypic flexibility may also permit a wide range of applications for stem cell research and processing.

Hepatogenesis of murine induced pluripotent stem cells in 3D micro-cavitary hydrogel system for liver regeneration.

The discovery of induced pluripotent stem cell (iPSC) technology has raised hopes in circumventing the current limitations in cell-based therapies where autologous stem cells could be generated from terminally differentiated somatic cells. Given the relatively short history of iPSC research, most of the studies are scientific exploratory in nature and hence have minimal practical usage. In this study, we aimed to combine existing knowledge on iPSC differentiation with three-dimensional (3D) scaffold platform so as to fabricate implantable constructs for liver regeneration. A micro-cavitary hydrogel (MCG) platform was employed as a continuous system for both colonies and/or EBs formation and differentiation. The advantage of MCG system is that it further enhances nutrient exchange and also permits greater living space for the encapsulated pluripotent stem cells to rapidly grow into colonies and/or EBs compared to typical non-MCG system. Murine iPSCs and embryonic stem cells (ESCs) were encapsulated respectively in alginate MCG system and after culturing for 10 days; colonies/EBs were formed spontaneously. Differentiation conditions were then introduced to direct the cells toward endodermal lineage and subsequently hepatic lineage and maturation. Up-regulation of endoderm markers and hepatic markers was observed in both iPSCs and ESCs suggesting that iPSC as effectively as the ESCs in MCG system. Urea and albumin production were significantly higher compared to monolayer culture, demonstrating the beneficial effects of MCG system. The results from this work provide foundation in understanding of iPSC differentiation in 3D engineered environment and aid in future biomedical research of iPSC technology.

Formation of model hepatocellular aggregates in a hydrogel scaffold using degradable genipin crosslinked gelatin microspheres as cell carriers

Primary hepatocyte is probably the preferred cell for cell therapy in liver regeneration. However, its non-ideal proliferation capacity and rapid loss of phenotype during 2D culture compromises the quality and quantity of the transplanted hepatocytes, resulting in variable success rates of this treatment. Many studies have shown that the formation of 3D hepatocellular spheroids aids in the maintenance of liver-specific functions in hepatocytes. However, many of the methodologies employed require a sophisticated set-up or specialized equipment which makes it uneconomical to scale up for clinical applications. In this study, we have developed dual-functioning genipin crosslinked gelatin microspheres that serve as cell carriers as well as porogens for delivering the model cells and also for creating cavities. The cells were first seeded onto genipin crosslinked gelatin microspheres for attachment, followed by encapsulation in alginate hydrogel. Collagenase, MMP-9, was introduced either in the culture media or mixed with alginate precursor solution to allow microsphere degradation for creating cavities within the gel bulk. Accordingly, the cells proliferate within the cavities, forming hepatocellular aggregates while the alginate hydrogel serves as a confinement, restricting the size and the shape of the aggregates to the size of the cavities. In addition, the final hepatocellular aggregates could be harvested from the system by removing the alginate hydrogel via citrate treatment. Therefore, this versatile platform not only has the advantage of injectability and simplicity, the cellular aggregates generated are in a controlled size and shape and can be extracted from the system.

Genipin-crosslinked microcarriers mediating hepatocellular aggregates formation and functionalities.

In engineered regenerative medicine, various types of scaffolds have been customized to pursue the optimal environment for different types of therapeutic cells. In liver therapeutic research, hepatocytes require attachment to solid anchors for survival and proliferation before they could grow into cellular aggregates with enhanced functionalities. Among the various biomaterials scaffolds and vehicles, microspherical cell carriers are suited to these requirements. Individual spheres may provide two-dimensional (2D) cell-affinitive surfaces for cell adhesion and spreading; whereas multiple microcarriers may form three-dimensional (3D) matrices with inter-spherical space for cell expansion and multicellular aggregation. In this study, we culture human liver carcinoma cell line (HepG2) cells on genipin-crosslinked gelatin microspheres of two different sizes. Results suggest that both microcarriers support cell adhesion, proliferation, and spontaneous formation of hepatocellular aggregates, among which the spheres with bigger size (200-300 μm) seem more favorable than the smaller ones in terms of aggregate formation and liver specific functionalities. These findings suggest that the genipin-crosslinked microcarrier is a competent vehicle for liver cell delivery.

Release of transgenic progranulin from a living hyaline cartilage graft model: An in vitro evaluation on anti-inflammation.

Osteoarthritis (OA) is a prevalent condition that compromises and even jeopardizes the life quality of millions of people. Common symptoms in OA includes joint stiffness and soreness, and they are often associated with inflammations to various extend. Due to the avascular and aneural nature of articular hyaline cartilage, it has limited self-repair capabilities; especially under inflammatory conditions, damages inflicted on cartilage are often irreversible. Hence, treatment approaches focus on anti-inflammation or articular cartilage replacement. In this study, an engineered, dual-functional living hyaline cartilage graft (LhCG), capable of releasing transgenic anti-inflammatory cytokine-progranulin (PGRN) is developed and envisioned to simultaneously fulfil both requirements. The therapeutic functionality of PGRN releasing LhCG is evaluated by co-culturing the constructs with tumor necrosis factor-alpha (TNFα) secreting THP-1 cells to simulate the inflammatory condition in arthritis. Non-transgenic LhCG constructs and non-coculture sample groups were set up as controls. Gene expression and ECM composition changes across samples were assessed to understand the effects of PGRN as well as inflammatory environment on the cartilage graft. Collectively, the results in this study suggest that in situ release of transgenic recombinant PGRN protects LhCG from induced inflammation in vitro; contrastively, in the absence of PGRN, cartilage grafts are at risk of being degraded and mineralized under exposure to TNFα signaling. This shows that cartilage graft itself can be at risk of degradation or calcification when implanted in arthritic microenvironment. Hence, the inflammatory microenvironment has to be considered in cartilage replacement therapy to increase chances of successful joint mobility restoration.

Use of Interim Scaffolding and Neotissue Development to Produce a Scaffold-Free Living Hyaline Cartilage Graft

The fabrication of three-dimensional (3D) constructs relies heavily on the use of biomaterial-based scaffolds. These are required as mechanical supports as well as to translate two-dimensional cultures to 3D cultures for clinical applications. Regardless of the choice of scaffold, timely degradation of scaffolds is difficult to achieve and undegraded scaffold material can lead to interference in further tissue development or morphogenesis. In cartilage tissue engineering, hydrogel is the highly preferred scaffold material as it shares many similar characteristics with native cartilaginous matrix. Hence, we employed gelatin microspheres as porogens to create a microcavitary alginate hydrogel as an interim scaffold to facilitate initial chondrocyte 3D culture and to establish a final scaffold-free living hyaline cartilaginous graft (LhCG) for cartilage tissue engineering.