Injectable hyaluronic acid scaffolds for cartilage tissue engineering

Abstract

Every year tens of millions worldwide suffer from cartilage damage, caused by mechanical degradation, trauma or disease. Because of the lack of blood supply and low cell concentration within the tissue, cartilage has very limited regenerative ability. Although current treatments can provide symptomatic relief, the results vary greatly among individuals, and newly formed tissue often does not duplicate the structure, composition or mechanical properties of normal cartilage. Therefore, in recent years, tissue engineering has emerged as an alternative therapy. Tissue engineering enhances the body's natural healing capacity by providing cells, signaling molecules, and an environment in the form of a scaffold that is conducive to tissue growth. This project has focused on the development of a tissue engineering scaffold for cartilage regeneration. Disadvantages to current scaffolds include the fact that they require surgery for implantation, and that they are difficult to mold to the exact shape of the defect site. Hence, the motivation of this thesis is to develop an injectable scaffold that can be administered in a minimally invasive manner, and that allows for scaffold formation in situ, naturally shaping the construct into the shape of the defect, and thus promoting integration and stability To this end, we have developed a thermoresponsive injectable scaffold for cartilage tissue engineering. The scaffold was injected as a liquid at room temperature, and gelled at the target site in response to the change to body temperature, resulting in a biocompatible, bioresorbable substrate for tissue growth. Our approach involved suspending thermoresponsive liposomes, which encapsulated a crosslinking agent, in a polymer solution. At room temperature, the crosslinking agent was separated from the polymer by the lipid membrane, hence the precursor solution remained a liquid and injectable. Upon injection and exposure to body temperature, the lipids experienced a phase transition, which significantly increased the membrane permeability and led to the release of the crosslinking agent and reaction with the polymer, forming a networked scaffold.

(cont.) The scaffold system that we have chosen is a hyaluronic acid-tyramine system (HA-Tyr) that crosslinked in the presence of H202 and horseradish peroxidase (HRP) to form a hydrogel. Since HA, Tyr, H202 and peroxidases all occur naturally in the body, scaffold formation could take place with minimal toxicity and in the presence of cells as well as in situ. In order to impart temperature sensitivity to this system, HRP was encapsulated within liposomes, and it was shown that HRP was successfully retained at 25°C and released at 37°C. Upon liposome addition to the HA-Tyr/H202 solution, the precursor solution remained a liquid for hours at 25°C, yet gelation could be induced within minutes when exposed to 37°C. Furthermore, it was shown that gelation times could be adjusted to meet various clinical needs by modulating HRP encapsulation, liposome concentration and HA-Tyr concentration. In order to test the potential of the HA-Tyr system for cartilage production, porcine chondrocytes were encapsulated within HA-Tyr/H202/HRP hydrogels and implanted subcutaneously in mice. Harvested constructs were shown to achieve a GAG content of 1.2 wt% and demonstrated 40% of the collagen content of normal articular cartilage. Matrix production was found to be influenced by the initial cell density, scaffold degradation rate and Type II collagen concentration. The means of HRP delivery, whether by simple addition or through thermoreponsive liposomes, was not shown to have an effect on matrix production. Injected scaffolds were shown to achieve GAG and collagen levels similar to that of implanted scaffolds. As signaling molecules have been demonstrated to be potent chondrogenic inducers, PLGA-hydroxyapatite nanocomposite microparticles were utilized for the controlled delivery of TGF-[beta]1 and IGF-1. The rate of growth factor release was modulated by the molecular weight of PLGA within the microparticles; increasing molecular weight led to decreasing release rate. The nanocomposite microparticles were encapsulated within HA-Tyr/H202/HRP/chondrocyte constructs, which were then implanted subcutaneously in mice.

(cont.) Growth factor-induced enhancement of GAG and collagen production was found to be determined by the release rates of TGF-31 and IGF-1, multifactor release, and the dosage of nanocomposite microparticles. Injection of the microparticles with an HA-Tyr/H202/HRP liposome/chondrocyte/collagen solution also showed that the microparticles did not interfere with in situ scaffold formation, and could induce significant improvements to GAG and collagen production in the injectable system.