Yen-Chih Lin

Synthesis and characterization of collagen/hyaluronan/chitosan composite sponges for potential biomedical applications

Cells, scaffolds and growth factors are three main components of a tissue-engineered construct. Collagen type I, a major protein of the extracellular matrix (ECM) in mammals, is a suitable scaffold material for regeneration. Another important constituent of the ECM, hyaluronic acid (hyaluronan, HA), has been used for medical purposes due to its hydrogel properties and biodegradability. Chitosan is a linear polysaccharide comprised of beta1- to beta4-linked d-glucosamine residues, and its potential as a biomaterial is based on its cationic nature and high charge density in solution. This study was conducted to evaluate the characteristics of scaffolds composed of different ratios of type I comb collagen and chitosan with added HA in order to obtain the optimum conditions for the manufacture of collagen-hyaluronan-chitosan (Col-HA-Ch; comprising collagen, HA and chitosan mixed in different ratios: 10:1:0, Col10HACh0; 9:1:1, Col9HACh1; 8:1:2, Col8HACh2; 7:1:3, Col7HACh3; 6:1:4, Col6HACh4; and 5:1:5, Col5HACh5) composite porous scaffolds. Microstructural observation of the composite scaffolds was performed using scanning electron microscopy. The mean pore diameters ranged from 120 to 182microm and decreased as the chitosan composition increased. All scaffolds showed high pore interconnectivity. Swelling ratio measurements showed that all specimens could bind 35- to 40-fold of physiological fluid and still maintain their form and stability. For tensile strength, the optimal ratio of collagen and chitosan was 9:1. Thermal stability was investigated using a differential scanning calorimeter and showed that Col5HACh5 and Col6HACh4 were significantly more stable than the other groups. In enzymatic sensitivity, a steady increase in the biostability of the scaffolds was achieved as the chitosan concentration was increased. In biocompatibility testing, the proliferation of the fibroblasts cultured in Co-HA-Ch tri-copolymer scaffolds was high. Overall, we observed the 9:1:1 mixing ratio of collagen, hyaluronan and chitosan to be optimal for the manufacture of complex scaffolds. Furthermore, Col-HA-Ch tri-polymer scaffolds, especially Col9HACh1, could be developed as a suitable scaffold material for tissue engineering applications.

Diffusion of soluble factors through degradable polymer nerve guides: Controlling manufacturing parameters.

Nerve guides are cylindrical conduits of either biologically based or synthetic materials that are used to bridge nerve defects. While it is well known that a critical aspect of nerve regeneration is the delivery of oxygen and nutrients to the surviving nerve tissue, several guide parameters that determine the permeability of nerve guides to nutrients are often overlooked. We have reproducibly manufactured poly(caprolactone) (PCL) nerve guides of tailored porosity percentage, wall thickness and pore diameter through a dip-coating/salt-leaching technique. In this study, these three parameters were varied to measure the response of glucose and lysozyme diffusion through the guide wall. In addition, nerve guide permeability following protein fouling studies was examined. Based on the results from this study, it was determined that at high porosity percentages (80%), decreasing the pore diameter (10-38microm) was a measurable method of decreasing the lysozyme permeability of PCL nerve guides while not creating a loss of glucose permeability. PCL fouling studies were used to fine-tune the desirable nerve guide wall thickness. Results indicated that nerve guides 0.6mm thick decreased the loss of lysozyme to almost 10% without significantly diminishing glucose (nutrient) permeability. These results will be utilized to optimize nerve guide parameters for future in vivo applications.

Peptide modification of polyethersulfone surfaces to improve adipose-derived stem cell adhesion.

Polyethersulfone (PES) is a nondegradable, biocompatible, synthetic polymer that is commonly utilized as a membrane material for applications such as hemodialysis, ultrafiltration and bioreactor technology. Various studies have shown surface modification to be a valuable tool in the development of nondegradable materials which promote cell adhesion. Cells of interest include adipose-derived stem cells (ASCs). ASCs are multipotent mesenchymal stem cells that are useful for various regenerative medicine applications. In this study, we hypothesized that PES surfaces modified with a peptide sequence based from fibronectin, such as Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser and Gly-Arg-Gly-Asp-Ser, would increase ASC adhesion compared to unmodified PES surfaces. The synthetic peptides were covalently bonded to amine-modified PES surfaces using 1-ethyl-3-(dimethylaminopropyl) carbodiimide. The surfaces were characterized using a ninhydrin assay and contact angle measurements. The ninhydrin assay confirmed the presence of amine groups on the surface of peptide-treated PES disks. Advancing water contact angles were analyzed to detect changes in the hydrophilicity of the polymer surfaces, and results indicated our PES membranes had excellent hydrophilicity. The attachment and proliferation of human ASCs was assessed and RGD-treated surfaces resulted in a higher number of attached ASCs after 6 and 48 h, as compared to unmodified PES surfaces. Additionally, varying concentrations of the RGD peptide sequence concentration were examined. These results indicate that PES membranes modified with the RGD peptide sequence can be utilized for enhanced ASC attachment in biomedical applications.

Injectable systems and implantable conduits for peripheral nerve repair

Acute sensory problems following peripheral nerve injury include pain and loss of sensation. Approximately 360,000 people in the United States suffer from upper extremity paralytic syndromes every year. Restoration of sufficient functional recovery after long-gap peripheral nerve damage remains a clinical challenge. Potential nerve repair therapies have increased in the past decade as the field of tissue engineering expands. The following review describes the use of biomaterials in nerve tissue engineering. Namely, the use of both synthetic and natural biomaterials, including non-degradable and degradable nerve grafts, is addressed. The enhancement of axonal regeneration can be achieved by further modification of the nerve guides. These approaches include injectable hydrogel fillers, controlled drug delivery systems, and cell incorporation. Hydrogels are a class of liquid-gel biomaterials with high water content. Injectable and gelling hydrogels can serve as growth factor delivery vehicles and cell carriers for tissue engineering applications. While natural hydrogels and polymers are suitable for short gap nerve repair, the use of polymers for relatively long gaps remains a clinical challenge.

Synergistic lithium chloride and glial cell line-derived neurotrophic factor delivery for peripheral nerve repair in a rodent sciatic nerve injury model.

Background: Restoring peripheral nerve function after long gap peripheral nerve damage is challenging. Lithium chloride has demonstrated neuroprotective qualities and therefore shows great potential therapeutic benefit for some neurodegenerative diseases. This study examined the synergistic combination of glial cell line–derived neurotrophic factor and lithium chloride and its effect on peripheral nerve regeneration in a rat sciatic nerve injury model. Methods: Polycaprolactone conduits with glial cell line–derived neurotrophic factor–loaded double-walled microspheres and local injections of lithium chloride, 1.5 or 2.5 mEq/kg body weight, were examined in a 15-mm rat sciatic nerve defect model. Eighteen Lewis male rats were divided randomly into control, 1.5-, and 2.5-mEq/kg lithium chloride injection groups. As an indicator of recovery, nerve sections were stained with S100, protein gene product 9.5 antibody, and toluidine blue. Results: Nerves stained with S100 and protein gene product 9.5 antibody demonstrated a significantly increased density of Schwann cells and axons in the 2.5-mEq/kg lithium chloride injection–treated groups compared with both the control and 1.5-mEq/kg lithium chloride injection–treated groups (p < 0.05). At 6 weeks, histomorphometry revealed a significantly higher fiber density in the middle of the conduit for the 2.5-mEq/kg groups compared with the 1.5-mEq/kg group or the control group. Conclusion: Polycaprolactone nerve guides with glial cell line–derived neurotrophic factor–loaded double-walled microspheres and local injections of lithium chloride, 2.5-mEq/kg, represent a potentially viable guiding material for Schwann cell and axon migration and proliferation for the treatment of peripheral nerve regeneration.

Analysis of type II diabetes mellitus adipose-derived stem cells for tissue engineering applications.

To address the functionality of diabetic adipose-derived stem cells in tissue engineering applications, adipose-derived stem cells isolated from patients with and without type II diabetes mellitus were cultured in bioreactor culture systems. The adipose-derived stem cells were differentiated into adipocytes and maintained as functional adipocytes. The bioreactor system utilizes a hollow fiber–based technology for three-dimensional perfusion of tissues in vitro, creating a model in which long-term culture of adipocytes is feasible, and providing a potential tool useful for drug discovery. Daily metabolic activity of the adipose-derived stem cells was analyzed within the medium recirculating throughout the bioreactor system. At experiment termination, tissues were extracted from bioreactors for immunohistological analyses in addition to gene and protein expression. Type II diabetic adipose-derived stem cells did not exhibit significantly different glucose consumption compared to adipose-derived stem cells from patients without type II diabetes (p > 0.05, N = 3). Expression of mature adipocyte genes was not significantly different between diabetic/non-diabetic groups (p > 0.05, N = 3). Protein expression of adipose tissue grown within all bioreactors was verified by Western blotting.The results from this small-scale study reveal adipose-derived stem cells from patients with type II diabetes when removed from diabetic environments behave metabolically similar to the same cells of non-diabetic patients when cultured in a three-dimensional perfusion bioreactor, suggesting that glucose transport across the adipocyte cell membrane, the hindrance of which being characteristic of type II diabetes, is dependent on environment. The presented observation describes a tissue-engineered tool for long-term cell culture and, following future adjustments to the culture environment and increased sample sizes, potentially for anti-diabetic drug testing.