Three-dimensional scaffolds for tissue engineering applications : role of porosity and pore size

Tissue engineering applications commonly encompass the use of three-dimensional (3D) scaffolds to provide a suitable microenvironment for the incorporation of cells or growth factors to regenerate damaged tissues or organs. These scaffolds serve to mimic the actual in vivo microenvironment where cells interact and behave according to the mechanical cues obtained from the surrounding 3D environment. Hence, the material properties of the scaffolds are vital in determining cellular response and fate. These 3D scaffolds are generally highly porous with interconnected pore networks to facilitate nutrient and oxygen diffusion and waste removal. This review focuses on the various fabrication techniques (e.g., conventional and rapid prototyping methods) that have been employed to fabricate 3D scaffolds of different pore sizes and porosity. The different pore size and porosity measurement methods will also be discussed. Scaffolds with graded porosity have also been studied for their ability to better represent the actual in vivo situation where cells are exposed to layers of different tissues with varying properties. In addition, the ability of pore size and porosity of scaffolds to direct cellular responses and alter the mechanical properties of scaffolds will be reviewed, followed by a look at nature's own scaffold, the extracellular matrix. Overall, the limitations of current scaffold fabrication approaches for tissue engineering applications and some novel and promising alternatives will be highlighted.Published versio

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 surfacemodified 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.Published Versio

Combinatorial effect of different alginate compositions, polycations, and gelling ions on microcapsule properties

Microencapsulation technology is commonly used to deliver cells and drugs for therapeutic applications. The encapsulation material has a direct influence over the properties of microcapsules and will eventually dictate the efficacy of this delivery system. In this study, the combinatory effect of different alginate compositions, polycations and gelling ions was investigated to determine their roles in affecting the properties of the microcapsules. A multifactorial relationship was found between the three factors, in which certain factors took priority over others in influencing the overall property of the microcapsules. As the size of the microcapsules was kept constant throughout the investigation, further insights into the role of fabrication parameters on microcapsules size were also obtained. From the results, poly-L-lysine-coated microcapsules fabricated from 40/60 sodium alginate and cross-linked with barium chloride were the most ideal for applications that require both good mechanical as well as diffusion properties

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

Chitosan microsphere scaffold tethered with RGD-conjugated poly(methacrylic acid) brushes as effective carriers for the endothelial cells

Endothelial cell–matrix interactions play a vital role in promoting vascularization of engineered tissues. The current study reports a facile and controllable method to develop a RGD peptide-functionalized chitosan microsphere scaffolds for rapid cell expansion of human umbilical vein endothelial cells (HUVECs). Functional poly(methacrylic acid) (PMAA) brushes are grafted from the chitosan microsphere surfaces via surface-initiated ATRP. Subsequent conjugation of RGD peptides on the pendent carboxyl groups of PMAA side chain is accomplished by carbodiimide chemistry to facilitate biocompatibility of the 3D CS scaffolding system. In vitro cell-loading assay of HUVECs exhibits a significant improvment of cell adhesion, spreading, and proliferation on the RGD peptide-immobilized CS microsphere surfaces

Poly(4-vinylaniline)-polyaniline bilayer-modified stainless steels for the mitigation of biocorrosion by sulfate-reducing bacteria (SRB) in seawater

A novel strategy by combination of surface-initiated atom transfer radical polymerization (ATRP) and in situ chemical oxidative graft polymerization was employed to tether stainless steel (SS) with poly(4-vinylaniline)-polyaniline (PVAn-PANI) bilayer coatings for mitigating biocorrosion by sulfate-reducing bacteria (SRB) in seawater. A trichlorosilane coupling agent was first immobilized on the SS surfaces to provide sulfonyl halide groups for surface-initiated ATRP of 4-VAn. A subsequent grafting of PANI onto the PVAn-grafted surface was accomplished by in situ chemical oxidative graft polymerization of aniline. The PVAn-PANI bilayer coatings were finally quaternized by hexylbromide to generate biocidal functionality. The so-synthesized SS surface was found to significantly reduce bacterial adhesion and biofilm formation. Electrochemical results revealed that the PVAn-PANI modified SS surface exhibited high resistance to biocorrosion by SRB. With the inherent anticorrosion capability and antibacterial properties of quaternized PVAn-PANI bilayers, the functionalized SS substrates are potentially useful to steel-based equipment under harsh marine environments

Redox-responsive mesoporous silica nanoparticles : a physiologically sensitive codelivery vehicle for siRNA and doxorubicin

Aims: Efficient siRNA/drug codelivery carriers can offer great promises to cancer treatment on account of synergistic effect provided from cancer-associated gene and anticancer drugs. In this work, a redox-responsive drug/siRNA codelivery vehicle based on mesoporous silica nanoparticles was fabricated to simultaneously deliver siRNA and doxorubicin (Dox) in vitro and in vivo. Results: The nanoparticle surface was functionalized with the adamantane (AD) units. Formation of stable host–guest complex between disulfide bond linked-AD and ethylenediamine-modified β-cyclodextrin is capable of fully blocking drugs inside the nanopores, while amino groups can complex with siRNA via electrostatic interaction. Relatively high concentration of glutathione in biophysical environment provides natural reducing agent to trigger drug/siRNA release by cleaving pre-introduced disulfide bonds. B-cell lymphoma 2 (Bcl-2) siRNA was codelivered to silence Bcl-2 protein expression in HeLa cells, resulting in enhanced chemotherapy efficacy in vitro. In vivo delivery experiment carried out in transgenic zebrafish larvae indicates that the delivery of Dox inhibits the development of choroid plexus in a dose-dependent manner, leading to successful decrease of green fluorescence protein transcription in choroid plexus. Reduction of liver tumor was also demonstrated after injection of Dox-loaded nanoparticles. Innovation: We successfully demonstrated that functional nanoparticles could serve as an efficient carrier for the delivery of Bcl-2 siRNA and Dox in HeLa cells and in transgenic zebrafish larvae, leading to enhanced therapeutic efficacy. Conclusion: Enhanced cytotoxicity caused by simultaneous delivery of Bcl-2 siRNA and Dox was observed in HeLa cells. Drug-loaded nanoparticles were internalized in vivo, inhibiting the development of choroid plexus and the progression of liver tumor.Published versio

Comparative Study of Adipose-Derived Stem Cells From Abdomen and Breast

Background: Abdominal tissue enriched with adipose-derived stem cells (ASCs) is often used in cell-assisted lipotransfer procedures for breast reconstruction. However, as the tissue microenvironment and stem cell niche play important roles in defining the characteristics of the resident cells, it is hypothesized that the stem cell population present in the donor abdominal tissue has dissimilar properties as compared with the cells in the recipient breast tissue, which may ultimately affect the long-term success of the graft. Methods: Adipose-derived stem cells were isolated from breast and abdominal fat tissues and characterized for mesenchymal-specific cell surface markers, and their population doubling, colony-forming capabilities, and proliferative properties were compared. The multilineage potential of both cell populations was also investigated. Results: Adipose-derived stem cells from both tissue sites were found to possess similar marker expression and multilineage differentiation potential. However, breast fat–derived ASCs were observed to have a higher self-renewal capability and an unstable population doubling as compared with abdominal fat-derived ASCs. Gene expression studies revealed that the breast fat–derived ASCs were predisposed to the osteogenic lineage and the abdominal fat–derived ASCs to the adipogenic lineage. Conclusions: Cells derived from both fat tissues possess different characteristics in terms of their growth kinetics and predisposition to the osteolineages and adipolineages. In particular, ASCs from the abdominal tissue appear to contribute to adipose tissue turnover, whereas ASCs from breast tissue, if used for cell-assisted fat grafting, may potentially be responsible for complications in fat grafting, such as oil cysts, calcifications, fat necrosis, and tumors.Accepted versio

Development of a miniaturized stimulation device for electrical stimulation of cells

Background: Directing cell behaviour using controllable, on-demand non-biochemical methods, such as electrical stimulation is an attractive area of research. While there exists much potential in exploring different modes of electrical stimulation and investigating a wider range of cellular phenomena that can arise from electrical stimulation, progress in this field has been slow. The reasons for this are that the stimulation techniques and customized setups utilized in past studies have not been standardized, and that current approaches to study such phenomena rely on low throughput platforms with restricted variability of waveform outputs.Results: Here, we first demonstrated how a variety of cellular responses can be elicited using different modes of DC and square waveform stimulation. Intracellular calcium levels were found to be elevated in the neuroblast cell line SH-SY5Y during stimulation with 5 V square waves and, stimulation with 150 mV/mm DC fields and 1.5 mA DC current resulted in polarization of protein kinase Akt in keratinocytes and elongation of endothelial cells, respectively. Next, a miniaturized stimulation device was developed with an integrated cell chamber array to output multiple discrete stimulation channels. A frequency dividing circuit implemented on the device provides a robust system to systematically study the effects of multiple output frequencies from a single input channel. Conclusion: We have shown the feasibility of directing cellular responses using various stimulation waveforms, and developed a modular stimulation device that allows for the investigation of multiple stimulation parameters, which previously had to be conducted with different discrete equipment or output channels. Such a device can potentially spur the development of other high throughput platforms for thorough investigation of electrical stimulation parameters on cellular responses.Published versio

A current-mode stimulator circuit with two-step charge balancing background calibration

Current-mode CMOS stimulation systems have offered unprecedented opportunities for accurate and high through put in-vitro and in-vivo physiological studies. As these circuits are in long term contact with living organisms, they must be flexible, safe and power efficient. Any mismatch in biphasic current pulses will result in charge imbalance, leading to tissue/cell damage. Therefore, it is the most important to maintain the balance of the charge injected and retracted by the anode and the cathode, respectively. This work first adjusts the body biasing voltage of the anode to match with the cathode current. It is robust, process-variation-aware and can reduce the imbalanced current to less than 1%. Second, any residue charge at the stimulation site is retracted only when it reaches a critical value. This process is performed in the background and thus does not disturb the front-end operation. Overall, it can achieve less than 0.4 nA DC error current and thus is a suitable candidate for long term stimulation applications