Hyaline Cartilage versus Fibrocartilage Formation within Mechanically Loaded Cartilage Constructs: In Vitro and In Vivo Studies

Articular cartilage is a type of hyaline cartilage and it is crucial for joint movements, but aging or injury can cause changes that result in pain and disability. Osteoarthritis (OA) is a major joint disease resulting from advanced cartilage degeneration. Current surgical treatments do not allow for regenerating cartilage similar to native hyaline cartilage. Cartilage tissue engineering (CTE) is a potential therapeutic approach where engineered constructs are deployed into damaged cartilage of the joint to stimulate the production of hyaline cartilage extracellular matrix (ECM). To this end, hydrogels have been widely studied and examined due to their ability to provide a three-dimensioanl (3D) environment for maintaining chondrogenic phenotype in chondrocytes. It has been reported that in static cultures, hydrogel constructs have shown promise in producing key markers of hyaline cartilage, including GAGs and Col2, while producing low levels of Col1 indicative of fibrocartilage formation. Notablly, previous studies in CTE, under dynamic or mechanical loading, have primarily focused on the production of Col2 and GAGs in hydrogels, lacking studiesor investigations into Col1 production both in vitro and in vivo. This raise a great need to understand the formation of Col1, which is essential to the success in CTE. In this thesis, it is hypothesized that cells produce fibrocartilage when subjected to mechanical forces. To test this hypothesis, the research presented in this thesis aimed to assess both hyaline cartilage and fibrocartilage formation within 3D-bioprinted constructs, which are subject to mechanical stimulation or loading both in vitro and in vivo, with the following three specific objectives (each for testing partially the forementioned hypothesis).
The first objective was to test if increased mechanical compression enhances fibrocartilage ECM production in hydrogel constructs. For this, hydrogel constructs were fabricated from alginate and ATDC5 cells (a well-established in vitro model for chondrogenesis) by means of 3D-bioprinting and then cultured under varying compressive strains of 6%, 12%, and 24%, respectively, to explore if increasing the compressive force would cause more fibrocartilage-like ECM formation. The results showed that fibrocartilage-like ECM production tended to decrease in hydrogels when the compressive force was increased from unloaded to 12% loaded condition. However, its production then was enhanced in a statistically significant way by increasing compressive strain from 12% to 24%. Also, cell population seemed to decrease in a non-significant way in the hydrogel constructs subjected to 12% and 24% compressive strains.
The second objective was to examine the effects of (1) compression duration or period and (2) reinforcement added to the hydrogel constructs on their performance. By varying the compression duration, the hydrogel constructs were examined in terms of retention of proliferating cells and production of cartilaginous ECM including GAGs and total collagen in addition to hyaline cartilage vs fibrocartilage specific ECM productions. Due to the limited 3D structureal integrity of consturcts made from pure hydrogel, the second part of this objective was rational to create hydrogle constructs, while being reinforced with 3D-printed poly-caprolactone (PCL) structures, and future to examine the effect of reinforcement on the consturct performance. The results demonstrated that a longer compression period did not increase cell population or ECM content of loaded unreinforced hydrogels in a statistically significant way, but reinforced constructs did and that with an extended period, the reinforced constructs increased the fibrocartilage formation.
The third objective was to test the hypothesis in vivo, where varying mechanical forces (including compression, tensile, and shear) exist as compared to in vitro stiumuation with a single-direction force. For this, both hydrogel and hybrid constructs were designed and printed, and then implanted simutanously at the same site of the knee joints of mature pigs. Hybrid constructs were proposed to play a force-shielding role owing to having synthetic PCL strands, which could shield the cell-impregnated alginate strands from the applied forces. The results from this study showed that hydrogel constructs did not promote fibrocartilage formation in vivo. Defects with implanted hydrogels have shown abundant Col2 deposition over the 3-months, accompanied by a satisfactory level of Col1 deposition, compared to the natural articular cartilage. This deposition was observed primarily at the superficial regions. On the other hand, the hybrid constructs were unsuccessful in promoting cartilage regeneration in vivo, and their force-shielding attributes did not contribute to the improvement of hyaline cartilage regeneration. The present study found that synchrotron-based (SR) inline-phase-contrast imaging microcomputed tomography (inline-PCI-CT) is a feasible substitute for safranin O histology to assess cartilage regeneration. Additionally, inline-PCI-CT was able to visualize implanted PCL strands, regenerated tissues within the defects, and surrounding bone and cartilage components, despite the fact that small-scale details of the structures like cells were not discernible on inline-PCI-CT slices compared to histology images.
Taken all together, this thesis illustrates how hyaline cartilage vs fibrocartilage formation along with cells growth and ECM content varies in hydrogels, both unreinforced and reinforced, in response to compressive culture. Furthermore, the in vivo study provides insights into the use of hydrogel and hybrid constructs for cartilage regeneration, and the applications of inline-PCI-CT for cartilage assessments.