Chemically modified collagen and associated naturally occurring biomaterials and their potential applications in tissue engineering

Abstract

Glycosaminoglycans (GAGs) represent an important extracellular matrix (ECM), particularly in GAG-rich tissues such as nucleus pulposus (NP) and cartilage. The relative abundance of GAGs is represented by the GAG/hydroxyproline (HYP) ratio, which is 27:1, 1.3-4.2:1, and <0.1 in young adult NP, cartilage, and bone, respectively. In common musculoskeletal diseases such as disc degeneration and osteoarthritis, typical pathophysiological changes include significant loss of GAG and reduction of GAG/HYP ratio, followed by loss of the normal function of tissues.

The fabrication of GAG-rich scaffolds is full of challenges as GAG is rapidly released due to GAG is highly hydrophilic, therefore, the GAG/HYP ratio of the existing scaffolds cannot be higher than 4.5:1, which is significantly lower than the native NP. In this study, we hypothesize that by chemically modifying certain ECM components, including collagen (Col) and hyaluronic acid (HA), novel biomaterials that mimic the complex ECM of GAG-rich tissues, and novel biomaterials that effectively deliver genes into cells will be synthesized. In Chapters 2 and 3, positively charged aminated collagen (aCol) was combined with aminated hyaluronic acid (aHA) to effectively incorporate GAGs into the ECM meshwork in synthesizing a compositionally, structurally, and functionally biomimetic ECM for GAG-rich tissues. In Chapter 4, positively charged aCol was combined with another naturally occurring biomaterial chitosan to effectively deliver genes and hence augment growth factor secretion of cells for tissue engineering application.

Chapter 2 reports the fabrication of a novel GAG-rich material, namely Aminated Collagen-Aminated Hyaluronic Acid-GAG (aCol-aHA-GAG), and its potential application in NP tissue engineering. Compositionally, the aCol-aHA-GAG showed a record high GAG/ HYP ratio up to 39.1:1 in a controllable manner. Structurally, the aCol-aHA-GAG recapitulated the characteristic ‘nanobeads’ ultrastructure in SEM and ‘bottlebrush’ ultrastructure in TEM, supported the viability of bNPCs, and maintained their morphology and phenotype markers. Mechanically, the reduced elastic modulus and disc height recovery of aCol-aHA-GAG was similar to that of the native NP.

Chapter 3 reports the potential application of a series of GAG-incorporated materials, including aCol-aHA-GAG, to support the multiple differentiation potential of stem cells. We compared the effects of these GAG-incorporated materials on the differentiation of human mesenchymal stem cells (hMSCs) into osteogenic, chondrogenic, and discogenic lineage. In osteogenic differentiation, the Col (GAG/HYP 0) scaffold showed higher calcium (Ca) and phosphorus (P) deposition and Ca/P ratio and osteogenic phenotypic expression. In chondrogenic differentiation, aCol-GAG (GAG/HYP 4.9:1) showed higher GAG deposition and higher chondrogenic phenotype expression. In discogenic differentiation, the aCol-aHA-GAG (GAG/HYP 19.8:1) showed intensive GAG deposition and higher phenotypic expression of NPCs.

Chapter 4 demonstrates the synthesis of a novel biomaterial-based non-viral transfection reagent, namely aminated collagen-Chitosan (aCol-Chi), for gene transfection and growth factor secretion. The aCol-Chi showed enhanced cellular uptake and gene transfection efficiency than Chi, and great potential in bone morphogenetic protein-2 (BMP2) secretion and osteogenic differentiation.

This work demonstrated that chemical modification of ECM components and associated novel biomaterials facilitated the incorporation of GAGs and condensation with plasmids, facilitating their potential applications in scaffolding, cell and molecular therapy for musculoskeletal tissue engineering