UC Davis

Tissue regeneration, once a dream or a fantasy, is now becoming a reality. Science has advanced so much that cells can be cryopreserved and expanded into millions under laboratory conditions. Cells can be manipulated to become something they were not before and can be coerced into forming actual tissues. The discovery of powerful growth factors and the ability to synthesize them, helps to achieve these goals. Our understanding of pathologic processes and aging also plays an essential role in our ability to regenerate tissues. Laboratory methods have advanced immensely, allowing more efficient and rapid progress in regenerative medicine. This thesis addresses the regenerative efforts of two types of tissues: cartilage and skin. These tissues have an essential function in our bodies, but their structure and biology are dramatically different. Yet the common denominator is that people can benefit greatly from learning more about the regeneration of these tissues.

Cartilage is a shock-absorbing tissue lining the ends of diarthrodial joints that aims to provide a frictionless surface for joint articulation. In disease, damage of articular cartilage is often accompanied by tissue thinning and an irreversible yet progressive degeneration. Cartilage’s innately poor regenerative capacity and progressively increasing demand for its regeneration have resulted in astonishing developments and achievements in the field of tissue engineering (TE) and regenerative therapies. However, despite advancements in cartilage tissue engineering and current clinical transplantation practices, the ability to replace deficient or damaged cartilage remains limited. In response, researchers are urgently trying to develop novel and clinically applicable solutions for the treatment of cartilage degenerative disorders.

The translation of cartilage tissue engineering practices is hindered by the lack of suitable cell sources and implant integration with surrounding native tissue. The lack of implant integration is further heightened within the joint environment, where constant motion and loading generate tensile, compressive, and shear forces. The lack of native tissue-graft integration and long-term efficacious cartilage implantation suggest that further exploration is needed to see if immunology is playing a role in the lack of tissue integration. In addition, the quality of cartilage tissue generated varies greatly with different cell sources. Cartilage replacement therapies primarily utilize chondrocytes (cartilage cells) as their main cell source for generating cartilage tissue. Moreover, the chondrocytes can be sourced from multiple anatomical locations. The current harvesting procedure to extract chondrocytes for cartilage tissue engineering can be quite limited and invasive. Therefore, the exploration of different cell sources or chondrocyte harvesting locations would be beneficial for improving cartilage tissue engineering techniques.

Another indicator of cartilage tissue quality is the mechanical durability of the tissue to withstand daily forces. A current limitation of cartilage tissue engineering practices is the inability to generate tissues of sufficient biomimicry to that of native tissue. Previous attempts in our laboratory have shown that tissue engineered cartilage is inferior to native tissue in terms biochemical composition and mechanical durability. Our laboratory has explored several applications and interventions to improve the quality of tissue engineered cartilage. This discrepancy between native tissue and tissue engineered cartilage is also due to our lack of understanding of cartilage biomechanics and limiting testing modalities.

Tissue regeneration is not limited to cartilage tissue. The demand for skin regeneration primarily aims to repair wounds caused from burns, infections, or other reconstructive surgeries. However, skin regenerative therapies are limited in the availability of sufficient autograft tissue. In addition, facilitating internal regeneration is difficult due to deficient vascularity within the wound. With this approach, wound closure may be delayed or improper healing. Improper healing could lead to scarring or deformity which could lead to mental adversities. Researchers may have found a speedy and miraculous therapy to facilitate wound closure and wound vascularization: fish skin bandages. The science of this therapy has yet to be explored in a comprehensive fashion until now.

Chapter 1 of this dissertation provides a concise review of cartilage immunology at the tissue and cellular level as it relates to immune privilege. It covers the current cartilage replacement therapies and how the lack of efficacy of each one could be explained by chondrocyte expression of the major histocompatibility complex. The chapter summarizes the current arguments and evidence supporting and refuting the immune privilege status of cartilage. The chapter also outlines the immunomodulatory mechanisms seen in other immune-privileged tissues and cancer. It concludes with a provision of how current methodologies to manipulate immune recognition could be beneficial to cartilage replacement therapies.

Chapter 2 presents a comprehensive study that accomplishes the following: 1) It explores the immunogenicity of chondrocytes. 2) It explores a novel strategy for evading immune detection of allogeneic chondrocytes through genetic editing. 3) It constructs genetically engineered immune-universal tissue-engineered cartilage constructs. 4) It evaluates the safety and efficacy of genetically engineered chondrocytes as a cell source for tissue-engineered cartilage implants.

Chapter 3 explores auricular chondrocytes as a cell source for cartilage tissue engineering. This chapter first outlines the current sources of cartilage tissue engineering and the disadvantages. It next uses previously published methods to construct cartilage tissue constructs from auricular chondrocytes. Upon completion, the tissue was evaluated mechanically and biochemically to determine its credibility as a sufficient as a cell source to construct durable cartilage tissue.

Chapter 4 explores the biomechanical properties of canine articular cartilage across age using an in-situ testing model. It provides extensive characterization of biochemical composition-mechanical function relationships of canine articular surfaces and presents a comparison of these values across different joints. Specifically, it reports the instantaneous modulus at 15% strain, thickness, collagen content, glycosaminoglycan content, cellularity, and histological changes across age. Also, this chapter provides an innovative, quantitative, and visual assessment of regional distribution of mechanical properties of cartilage using 3D mapping of each surface. This chapter suggests that additional unknown factors may play a role in influencing cartilage’s mechanical properties.

Chapter 5 explored the feasibility of using fish skin bandages as a therapeutic option for third-degree burns. The study induces third-degree burns on mice and treats the burns with either tilapia-skin or hydrocolloid adhesive bandages. The study explored the therapeutic efficacy of fish-skin bandages vs hydrocolloid adhesive bandages at the histologic, hematologic, molecular, and gross level. This study may seem out of place with the other chapters. However, this chapter reflects on how to induce internal tissue regeneration using external therapeutics. By studying the healing mechanisms behind tilapia skin, perhaps these factors can be applied to cartilage and induce tissue regeneration in vivo.

In summary, this dissertation delineates the cumulative progression of studies providing robust techniques methodologies for tissue regeneration and durability following transplantation. This work provides a solid foundation for future assessment directed at full tissue replacement conditions. Furthermore, this platform can be optimized for regeneration of other tissue structures, such as skin.