Andrew C.A. Wan

The effect of matrix stiffness on mesenchymal stem cell differentiation in a 3D thixotropic gel

Recent studies have demonstrated the effect of matrix stiffness on the phenotype and differentiation pathway of mesenchymal stem cells (MSCs). MSCs differentiated into neural, myogenic or osteogenic phenotypes depending on whether they were cultured on two-dimensional (2D) substrates of elastic moduli in the lower (0.1-1 kPa), intermediate (8-17 kPa) or higher ranges (34 kPa). In this study, MSCs were cultured in thixotropic gels of varying rheological properties, and similar results were found for the three-dimensional (3D) culture as for the previous findings in 2D culture. For the 3D cell cultures in thixotropic gels, the liquefaction stress (tau(y)), the minimum shear stress required to liquefy the gel, was used to characterize the matrix stiffness. The highest expressions of neural (ENO2), myogenic (MYOG) and osteogenic (Runx2, OC) transcription factors were obtained for gels with tau(y) of 7, 25 and 75 Pa, respectively. Immobilization of the cell-adhesion peptide, RGD, promoted both proliferation and differentiation of MSCs, especially for the case of the stiffer gels (>75 Pa). This study demonstrated the usefulness of thixotropic gels for 3D cell culture studies, as well as the use of tau(y) as an effective measure of matrix stiffness that could be correlated to MSC differentiation.

Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits.

Prolonged delivery of neurotrophic proteins to the target tissue is valuable in the treatment of various disorders of the nervous system. We have tested in this study whether sustained release of nerve growth factor (NGF) within nerve guide conduits (NGCs), a device used to repair injured nerves, would augment peripheral nerve regeneration. NGF-containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site-specific delivery of NGF. The conduits were used to bridge a 10 mm gap in a rat sciatic nerve model. Three months after implantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower G-ratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, as compared with those from control conduits loaded with either saline alone, BSA microspheres, or NGF protein without microencapsulation. Beneficial effects on fiber diameter, G-ratio and fiber density were also observed in the permeable PPE NGCs. Thus, the results confirm a long-term promoting effect of exogenous NGF on morphological regeneration of peripheral nerves. The tissue-engineering approach reported in this study of incorporation of a microsphere protein release system into NGCs holds potential for improved functional recovery in patients whose injured nerves are reconstructed by entubulation.

A new nerve guide conduit material composed of a biodegradable poly(phosphoester)

There is a resurgence of interest in the development of degradable and biocompatible polymers for fabrication of nerve guide conduits (NGCs) in recent years. Poly(phosphoester) (PPE) polymers are among the attractive candidates in this context, in view of their high biocompatibility, adjustable biodegradability, flexibility in coupling fragile biomolecules under physiological conditions and a wide variety of physicochemical properties. The feasibility of using a biodegradable PPE, P(BHET-EOP/TC), as a novel NGC material was investigated. Two types of conduits were fabricated by using two batches of P(BHET-EOP/TC) with different weight-average molecular weights (Mw) and polydispersity indexes (PI). The polymers as well as conduits were non-toxic to all six types of cells tested, including primary neurones and neuronally differentiated PC12 cells. After in situ implantation in the sciatic nerve of the rat, two types of conduits triggered a similar tissue response, inducing the formation of a thin tissue capsule composed of approximately eight layers of fibroblasts surrounding the conduits at 3 months. Biological performances of the conduits were examined in the rat sciatic nerve model with a 10 mm gap. Although tube fragmentation, even tube breakage, was observed within less than 5 days post-implantation, successful regeneration through the gap occurred in both types of conduits, with four out of 10 in the Type I conduits (Mw 14,900 and PI 2.57) and 11 out of 12 in the Type II conduits (Mw 18,900 and PI 1.72). The degradation of conduits was further evidenced by increased roughness on the tube surface in vivo under scanning electron microscope and a mass decrease in a time-dependent manner in vitro. The Mw of the polymers dropped 33 and 24% in the Type I and II conduits, respectively, in vitro within 3 months. Among their advantages over other biodegradable NGCs, the PPE conduits showed negligible swelling and no crystallisation after implantation. Thus, these PPE conduits can be effective aids for nerve regeneration with potential to be further developed into more sophisticated NGCs that have better control of the conduit micro-environment for improved nerve regeneration.

Multi-layered microcapsules for cell encapsulation.

Mechanical stability, complete encapsulation, selective permeability, and suitable extra-cellular microenvironment, are the major considerations in designing microcapsules for cell encapsulation. We have developed four types of multi-layered microcapsules that allow selective optimization of these parameters. Primary hepatocytes were used as model cells to test these different microcapsule configurations. Type-1 microcapsules with an average diameter of 400 microm were formed by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 microm. Cells in these microcapsules exhibited improved cellular functions over those cultured on collagen monolayers. Type-II microcapsules were formed by encapsulating the Type-I microcapsules in another 2-5 microm ter-polymer shell and a approximately 5 microm collagen layer between the two ter-polymer shells to ensure complete cell encapsulation. Type-II microcapsules comprised of a macro-porous exoskeleton with materials such as alumina sol-gel coated on the Type-I microcapsules. Nano-indendation assay indicated an improved mechanical stability over the Type-I microcapsules. Type-IV microcapsules were created by encapsulating Type-III microcapsules in another 2-5 microm ter-polymer shell, with the aim of imparting a negatively charged smooth surface to minimize plasma protein absorption and ensure complete cell encapsulation. The permeability for nutrient exchange, cellular functions in terms of urea production and mechanical stability of the microcapsules were characterized. The advantages and limitations of these microcapsules for tissue engineering are discussed.

Lineage restricted progenitors for the repopulation of decellularized heart.

The severe shortage of available donor hearts necessitates the development of other options for heart replacement. Recent results underline the promise of the decellularized organ approach in engineering a functional heart. However, little is known so far regarding the ability of decellularized heart ECM to differentiate embryonic stem cells or committed progenitor cells. In the present work, we compared the differentiation potential of human embryonic stem cells (hESCs) and human mesendodermal cells (hMECs) derived from hESCs, in decellularized hearts under static culture. Expression of various cardiac specific markers such as cTnT, Nkx-2.5, Myl2, Myl7, Myh6 and CD31 was elucidated by gene expression, immunostaining and flow cytometry. Both hMECs and hESCs upregulated expression of cardiac markers upon differentiation, but they exclusively expressed genes for myosin light chain (Myl2, Myl7) and myosin heavy chain (Myh6), respectively. To enhance the differentiation ability of the stem/progenitor cells in the acellular constructs, they were implanted subcutaneously in SCID mice. Immunostaining of the explants revealed the persistence of cardiac marker expressing cells, but which lacked beating function. Our results indicate that the intact extracellular matrix components and preserved mechanical properties of the decellularized heart had directed differentiation of the stem/progenitor cells into the cardiac lineage.

A thixotropic nanocomposite gel for three-dimensional cell culture

Thixotropic materials, which become less viscous under stress and return to their original state when stress is removed, have been used to deliver gel-cell constructs and therapeutic agents. Here we show that a polymer-silica nanocomposite thixotropic gel can be used as a three-dimensional cell culture material. The gel liquefies when vortexed--allowing cells and biological components to be added--and resolidifies to trap the components when the shear force from spinning is removed. Good permeability of nutrients and gases through the gel allows various cell types to proliferate and be viable for up to three weeks. Human mesenchymal stem cells cultured in stiffer gels developed bone-like behaviour, showing that the rheological properties of the gel can control cell differentiation. No enzymatic, chemical, or photo-crosslinking, changes in ionic strength or temperature are required to form or liquefy the gel, offering a way to sub-culture cells without using trypsin-a protease commonly used in traditional cell culture techniques.

Preparation of a chitin-apatite composite by in situ precipitation onto porous chitin scaffolds.

Composites of chitin with calcium phosphate were obtained by in situ precipitation of the mineral from a supersaturated solution onto chitin scaffolds. The chitin scaffolds were obtained by freeze drying to give a highly porous structure possessing a polar surface favorable for apatite nucleation and growth. THe extent and arrangement of calcium phosphate deposits on the chitin and substituted chitin scaffolds were explored. Up to 55% by mass of calcium phosphate could be incorporated into chitin scaffolds. Deposits on the chitin surface were a continuous apatite carpet nature while deposits on carboxymethylated chitin surfaces displayed a spherical morphology. Carboxymethylation of chitin exerts an overall inhibitory effect towards calcium phosphate deposition, but it provides for site-specific nucleation of the mineral phase. In situ precipitation can be an important route in the future production of various polymer-calcium phosphate composites.

Nanomaterials for in situ cell delivery and tissue regeneration

Nanomaterials can be defined as materials that possess clearly defined features of less than 100nm, and whose nanostructured features confer characteristics crucial to the materials bulk property. The nanostructured features can be an intermediate or final state of the material in its synthesis process. The field of nanomaterials as applied to in situ cell delivery and tissue engineering is rapidly expanding. Nanomaterials that include peptide amphiphiles, self-assembling peptides, electrospun scaffolds, layer-by-layer complexes, nanotubes and nanocomposites have been applied to cell culture, encapsulation and delivery with promising results. As compared to scaffold-free cell delivery, nanomaterials are advantageous in terms of providing a means to control the biochemical and mechanical microenvironment of the cells. Nanomaterials are amenable to a bottom-up approach in functionalization and mechanical tuning, as illustrated in the examples presented in this review. Furthermore, nanomaterials such as DNA polyplexes and carbon nanotubes can also be incorporated into the cell delivery vehicle to improve the regenerative outcome. Lastly, while nanomaterials harbor much potential for cell delivery and tissue regeneration, further characterization is required in terms of clinical safety before these materials can be employed towards therapeutic applications.

Fabrication of poly(phosphoester) nerve guides by immersion precipitation and the control of porosity.

Immersion precipitation was employed as a method for the fabrication of polymeric conduits from P(BHET-EOP/TC), a poly(phosphoester) with an ethylene terephthalate backbone, to be applied as guidance channels for nerve regeneration. Coatings of various porosities could be obtained by immersing mandrels coated with a solution of the polymer in chloroform into non-solvent immersion baths, followed by freeze or vacuum-drying. The porosity of the coatings decreased with an increase in polymer molecular weight, drying time before precipitation and concentration of polymer solution. The effects of these parameters can be rationalized by employing ternary phase diagrams, where porosity is directly related to the degree of phase separation available to the system before gelation occurs. To afford improved porosity control, a new system was developed which employed the contrasting phase-separation behavior of P(BHET-EOP/TC)/chloroform solution in methanol and water. As water is essentially a non-solvent for the polymer, the demixing boundary of the P(BHET-EOP/TC)-CHCl3-H2O system is located close to the polymer-solvent edge of the phase diagram, while that of the P(BHET-EOP/TC)-CHCl3-MeOH system is located further away. A mixture of methanol and water allows the demixing boundary to be shifted to intermediate coordinates. By immersing P(BHET-EOP/TC) coatings in immersion baths containing different ratios of water and methanol, then gradually titrating the bath with methanol to a concentration of 70% (v/v) methanol, surface porosities ranging from 2 to 58% could be achieved.

Patterned prevascularised tissue constructs by assembly of polyelectrolyte hydrogel fibres

The in vivo efficacy of engineered tissue constructs depends largely on their integration with the host vasculature. Prevascularisation has been noted to facilitate integration of the constructs via anastomosis of preformed microvascular networks. Here we report a technique to fabricate aligned, spatially defined prevascularised tissue constructs with endothelial vessels by assembling individually tailored cell-laden polyelectrolyte hydrogel fibres. Stable, aligned endothelial vessels form in vitro within these constructs in 24 h, and these vessels anastomose with the host circulation in a mouse subcutaneous model. We create vascularised adipose and hepatic tissues by co-patterning the respective cell types with the preformed endothelial vessels. Our study indicates that the formation of aligned endothelial vessels in a hydrogel is an efficient prevascularisation approach in the engineering of tissue constructs.