Casey K. Chan

Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin

Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(L-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.

The fabrication of nano-hydroxyapatite on PLGA and PLGA/collagen nanofibrous composite scaffolds and their effects in osteoblastic behavior for bone tissue engineering

Bone is a nanocomposite consisting of two main components, nano-hydroxyapatite (n-HA) and Type I collagen (Col). The aim is to exploit the nano-scale functional and material characteristics of natural bone in order to modulate cellular functions for optimal bone repair in bone graft systems. Here, we present an effective and novel technique in obtaining n-HA in cognate with native apatite on electrospun nanofibers within minutes without any pre-treatment. Using an alternate calcium and phosphate (Ca-P) solution dipping method, n-HA was formed on poly(lactide-co-glycolide) acid (PLGA) and blended PLGA/Col nanofibers. The presence of the functional groups of collagen significantly hastened n-HA deposition closed to nine-fold. The quantity of n-HA impinged upon the specific surface area, whereby mineralized PLGA/Col had a greater surface area than non-mineralized PLGA/Col, whereas n-HA did not significantly improve the specific surface area of mineralized PLGA compared to pure PLGA. The novelty of the process was that n-HA on PLGA had a positive modulation on early osteoblast capture (within minutes) compared to pure PLGA. Contrary, cell capture on mineralized PLGA/Col was comparable to pure PLGA/Col. Interestingly, although n-HA impeded proliferation during the culture period (days 1, 4 and 7), the cell functionality such as alkaline phosphatase (ALP) and protein expressions were ameliorated on mineralized nanofibers. The amount of n-HA appeared to have a greater effect on the early stages of osteoblast behavior (cell attachment and proliferation) rather than the immediate/late stages (proliferation and differentiation).

Biomimetic electrospun nanofibers for tissue regeneration

Nanofibers exist widely in human tissue with different patterns. Electrospinning nanotechnology has recently gained a new impetus due to the introduction of the concept of biomimetic nanofibers for tissue regeneration. The advanced electrospinning technique is a promising method to fabricate a controllable continuous nanofiber scaffold similar to the natural extracellular matrix. Thus, the biomedical field has become a significant possible application field of electrospun fibers. Although electrospinning has developed rapidly over the past few years, electrospun nanofibers are still at a premature research stage. Further comprehensive and deep studies on electrospun nanofibers are essential for promoting their biomedical applications. Current electrospun fiber materials include natural polymers, synthetic polymers and inorganic substances. This review briefly describes several typically electrospun nanofiber materials or composites that have great potential for tissue regeneration, and describes their fabrication, advantages, drawbacks and future prospects.

Electrospun Biocomposite Nanofibrous Scaffolds for Neural Tissue Engineering

Bridging of nerve gaps after injury is a major problem in peripheral nerve regeneration. Considering the potential application of a bio-artificial nerve guide material, polycaprolactone (PCL)/chitosan nanofibrous scaffolds was designed and evaluated in vitro using rat Schwann cells (RT4-D6P2T) for nerve tissue engineering. PCL, chitosan, and PCL/chitosan nanofibers with average fiber diameters of 630, 450, and 190 nm, respectively, were fabricated using an electrospinning process. The surface chemistry of the fabricated nanofibers was determined using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Simple blending of PCL with chitosan proved an easy and efficient method for fabricating PCL/chitosan nanofibrous scaffolds, whose surface characteristics proved more hydrophilic than PCL nanofibers. Evaluation of mechanical properties showed that the Youngs modulus and strain at break of the electrospun PCL/chitosan nanofibers were better than those of the chitosan nanofibers. Results of cell proliferation studies on nanofibrous scaffolds using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay showed 48% more cell proliferation on PCL/chitosan scaffolds than on PCL scaffolds after 8 days of culture. PCL/chitosan scaffolds showed better cell proliferation than PCL scaffolds and maintained their characteristic cell morphology, with spreading bipolar elongations to the nanofibrous substrates. This electrospun nanofibrous matrix thus proved of specific interest in tissue engineering for peripheral nerve regeneration.

STEM CELL HOMING IN MUSCULOSKELETAL INJURY

The regenerative potential of injured adult tissue suggests the physiological existence of cells capable of participating in the reparative process. Recent studies indicate that stem-like cells residing in tissues contribute to tissue repair and are replenished by precursor bone marrow-derived cells. Mesenchymal stromal cells (MSC) are among the candidates for reparative cells. These cells can potentially be mobilized into the circulation in response to injury signals and exert their reparative effects at the site of injury. Current therapies for musculoskeletal injuries pose unavoidable risks which can impede full recovery. Trafficking of MSC to the injury site and their subsequent participation in the regenerative process is thought to be a natural healing response that can be imitated or augmented by enhancing the endogenous MSC pool with exogenously administered MSC. Therefore, a promising alternative to the existing strategies employed in the treatment of musculoskeletal injuries is to reinforce the inherent reparative capacity of the body by delivering MSC harvested from the patients own tissues to the site of injury. The aim of this review is to inform the reader of studies that have evaluated the intrinsic homing and regenerative abilities of MSC, with particular emphasis on the repair of musculoskeletal injuries. Research that supports the direct use of MSC (without in vitro differentiation into tissue-specific cells) will also be reported. Based on accruing evidence that the natural healing mechanism involves the recruitment of MSC and their subsequent reparative actions at the site of injury, as well as documented therapeutic response after the exogenous administration of MSC, the feasibility of the emerging strategy of instant stem-cell therapy will be proposed.

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.

Synergistic effects of electrospun PLLA fiber dimension and pattern on neonatal mouse cerebellum C17.2 stem cells.

Topographical features, including fiber dimensions and pattern, are important aspects in developing fibrous scaffolds for tissue engineering. In this study aligned poly(l-lactide) (PLLA) fibers with diameters of 307+/-47, 500+/-53, 679+/-72 and 917+/-84 nm and random fibers with diameters of 327+/-40, 545+/-54, 746+/-82 and 1150+/-109 nm were obtained by optimizing the electrospinning parameters. We cultured neonatal mouse cerebellum C17.2 cells on the PLLA fibers. These neural stem cells (NSCs) exhibited significantly different growth and differentiation depending upon fiber dimension and pattern. On aligned fibers cell viability and proliferation was best on 500 nm fibers, and reduced on smaller or larger fibers. However, on random fibers cell viability and proliferation was best with the smallest (350 nm) and largest (1150 nm) diameter fibers. Polarized and elongated cells were orientated along the fiber direction on the aligned fibers, with focal contacts bridging the cell body and aligned fibers. Cells of spindle and polygonal morphologies were randomly distributed on the random fibers, with no focal contacts observed. Moreover, longer neurites were obtained on the aligned fibers than random fibers within the same diameter range. Thus, the surface topographic morphologies of fibrous scaffolds, including fiber pattern, dimensions and mesh size, play roles in regulating the viability, proliferation and neurite outgrowth of NSCs. Nevertheless, our results indicated that aligned 500 nm fiber are most promising for fine tuning the design of a nerve scaffold.

Processing nanoengineered scaffolds through electrospinning and mineralization suitable for biomimetic bone tissue engineering.

Processing scaffolds that mimic the extracellular matrix (ECM) of natural bone in structure and chemical composition is a potential promising option for engineering physiologically functional bone tissue. In this article, we report a novel method, by combining electrospinning and mineralization, to process a series of nano-fibrous scaffolding systems with desirable characteristics suitable for biomimetic bone tissue engineering. We have chosen two types of polymers, namely collagen and poly (lactic-co-glycolic acid) (PLGA), natural and synthetic of its kind, respectively, to electrospin into nano-fibrous scaffolds. The electrospun scaffolds have high surface area, high porosity and well connected open pore network. In order to mimic the chemical composition of native bone ECM, the electrospun scaffolds were subjected to mineralization under optimal conditions. From the experimental results, we observed that the formation of bone-like apatite into collagen was relatively abundant and significantly more uniform than PLGA. The major finding of this study has suggested that the surface functional groups of the scaffolding material, such as carboxyl and carbonyl groups of collagen, are important for the mineralization in vitro. In addition, this study revealed that the mineralization process predominantly induce the formation of nanosize carbonated hydroxyapatite (CHA) during collagen mineralization, whilst nanosize hydroxyapatite (HA) is formed during PLGA mineralization. These findings are critically important while selecting the material for processing bone scaffolding system.

Cell therapy for bone regeneration—Bench to bedside

The concept of bone tissue engineering, which began in the early 1980s, has seen tremendous growth in the numbers of research studies. One of the key areas of research has been in the field of mesenchymal stem cells, where the challenge is to produce the perfect tissue-engineered bone construct. This practical review summarizes basic and applied state-of-the-art research in the area of mesenchymal stem cells, and highlights the important translational research that has already been initiated. The topics that will be covered include the sources of stem cells in use, scaffolds, gene therapy, clinical applications in nonunions, tumors, osteonecrosis, revision arthroplasties, and spine fusion. Although significant challenges remain, there exists an exceptional opportunity to translate basic research in mesenchymal stem cell technologies into viable clinical treatments for bone regeneration.

Fabrication of Mineralized Polymeric Nanofibrous Composites for Bone Graft Materials

Poly-L-lactic acid (PLLA) and PLLA/collagen (50% PLLA+50% collagen; PLLA/Col) nanofibers were fabricated using electrospinning. Mineralization of these nanofibers was processed using a modified alternating soaking method. The structural properties and morphologies of mineralized PLLA and PLLA/Col nanofibers were investigated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and contact angle measurements. Human bone-derived osteoblasts were cultured on the materials for up to 1 week to assess the biological properties of the nanofibrous composites. Cell attachment on these nanocomposites was also tested within 1 h of culture at room temperature. The mechanical properties of the cell-nanocomposite constructs were determined using tensile testing. From our results, the bone-like nano-hydroxyapatite (n-HA) was successfully deposited on the PLLA and PLLA/Col nanofibers. We observed that the formation of n-HA on PLLA/Col nanofibers was faster and significantly more uniform than on pure PLLA nanofibers. The n-HA significantly improved the hydrophilicity of PLLA/Col nanofibers. From the results of cell attachment studies, n-HA deposition enhanced the cell capture efficacy at the 20-minute time point for PLLA nanofibers. The E-modulus values for PLLA+n-HA with cells (day 1 and day 4) were significantly higher than for PLLA+n-HA without cells. Based on these observations, we have demonstrated that n-HA deposition on nanofibers is a promising strategy for early cell capture.