Browsing by Subject "Chondrogenesis"

Osteoarthritis (OA) is one of the leading causes of disability in the United States, afflicting over 27 million Americans and imposing an economic burden of more than $128 billion each year (1, 2). OA is characterized by progressive degeneration of articular cartilage together with sub-chondral bone remodeling and synovial joint inflammation. Currently, OA treatments are limited, and inadequate to restore the joint to its full functionality.

Over the years, progresses have been made to create biologic cartilage substitutes. However, the repair of degenerated cartilage remains challenging due to its complex architecture and limited capability to integrate with surrounding tissues. Hence, there exists a need to create not only functional chondral constructs, but functional osteochondral constructs, which could potentially enhance affixing properties of cartilage implants utilizing the underlying bone. Furthermore, the molecular mechanisms driving chondrogenesis are still not fully understood. Therefore, detailed transcriptomic profiling would bring forth the progression of not only genes, but gene entities and networks that orchestrate this process.

Bone-marrow derived mesenchymal stem cells (MSCs) are routinely utilized to create cartilage constructs in vitro for the study of chondrogenesis. In this work, we set out to examine the underlying mechanisms of these cells, as well as the intricate gene correlation networks over the time course of lineage development. We first asked the question of how transforming growth factors are determining MSC differentiation, and subsequently utilized genetic engineering to manipulate this pathway to create an osteochondral construct. Next, we performed high-throughput next-generation sequencing to profile the dynamics of MSC transcriptomes over the time course of chondrogenesis. Bioinformatics analyses of these big data have yielded a multitude of information: the chondrogenic functional module, the associated gene ontologies, and finally the elucidation of GRASLND and its crucial function in chondrogenesis. We extended our results with a detailed molecular characterization of GRASLND and its underlying mechanisms. We showed that GRASLND could enhance chondrogenesis, and thus proposed its therapeutic use in cartilage tissue engineering as well as in the treatment of OA.

The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. In this dissertation, we demonstrate robust cartilaginous matrix production in multiple hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified chondroprogenitors demonstrated an improved chondrogenic capacity compared to unselected populations, improved matrix homogeneity, and reduced variability between tissues. We next demonstrated the ability of the system to serve as a high-throughput system for arthritis disease modeling using cytokine stimuli. Finally, we used this platform to screen for transcription factors whose activation might be involved in chondrogenic lineage specification of hiPSCs. Taken together, these studies describe the generation of a high-throughput system for chondrogenesis and its application for screens and arthritis disease modeling. Future applications of this platform may be useful for identifying pathways regulating cartilage regeneration and novel therapeutics for arthritis.

Articular cartilage is the thin, load-bearing connective tissue that lines the ends of long bones in diarthroidal joints, providing predominantly a mechanical function. Because cartilage is avascular and aneural, it has little capacity for self-repair if damaged. One repair strategy is through a functional tissue engineering approach using adipose-derived stem cells (ASCs). ASCs are an abundant progenitor cell source easily obtained through a minimally invasive liposuction procedure. When appropriately stimulated, ASCs have demonstrated significant potential for chondrogenic differentiation. Though studies have demonstrated the ability of ASCs to synthesize cartilage-specific macromolecules, a more thorough understanding of factors that modulate ASC chondrogenesis is required. Accordingly, the central aim of this dissertation was to study the chondrogenic response of ASCs to biochemical, biomechanical, and biomaterial factors.

We hypothesized that factors, other than TGF-beta and dexamethasone, would improve ASC chondrogenesis. BMP-6 emerged as a potent regulator of ASC chondrogenesis, particularly in early culture, as noted by significant upregulation of cartilage-specific extracellular matrix (ECM) genes and downregulation of cartilage hypertrophy markers.

Hypothesizing that biomechanical factors would accelerate the formation of cartilage-specific macromolecules, we designed and manufactured an instrument to apply dynamic deformational loading to ASC seeded constructs. Dynamic loading significantly inhibited ASC metabolism and downregulated cartilage-specific ECM genes. However, 21 days of dynamic loading induced the production of type II collagen, a principal component of articular cartilage.

We hypothesized that a biomaterial derived from cartilage would serve as a bioactive scaffold and induce chondrogenic differentiation. The novel, ECM-derived scaffold promoted the most robust differentiation of ASCs relative to both biochemical and biomechanical factors, particularly noted by a type II collagen-rich matrix after 28 days of culture. After 42 days of culture, biphasic mechanical testing revealed an aggregate modulus of 150 kPa, approaching that of native cartilage. These data suggest that the ECM-derived scaffold may retain important signaling molecules to drive differentiation or that ASC differentiation is dependent on proper cell anchorage.

In summary, we have shown that biochemical, biomechanical, and biomaterial factors have strong influences on the chondrogenic potential of ASCs. Optimization of these factors will ultimately be required to successfully engineer a functional tissue.

Articular cartilage is a smooth connective tissue that covers the ends of bones and protects joints from wear. Cartilage has a poor healing capacity, and the lack of treatment options motivates the development of tissue engineering strategies. The widespread cartilage degeneration associated with osteoarthritis (OA) is dramatically accelerated by joint injury, but the defined initiating event presents a therapeutic window for preventive treatments. In vitro model systems allow investigation of OA risk factors and screening of potential therapeutics. This dissertation develops stem-cell based strategies to 1) treat cartilage injury and OA using tissue-engineered cartilage, 2) prevent the development of OA by delivering stem cells to the joint after injury, and 3) study cartilage by establishing systems to model genetic and environmental contributors to OA.

Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are promising human adult cell sources for cartilage tissue engineering, but require distinct chondrogenic conditions. As compared to ASCs, MSCs demonstrated enhanced chondrogenesis in both alginate beads and cartilage-derived matrix scaffolds.

We hypothesized that MSC therapy would prevent post-traumatic arthritis (PTA) by altering the balance of inflammation and regeneration. Highly purified MSCs (CD45-TER119-PDGFRα+Sca-1+) rapidly expanded under hypoxic conditions. Unexpectedly, MSCs from control C57BL/6 (B6) mice proliferated and differentiated more than MSCs from MRL/MpJ (MRL) "superhealer" mice. We injected B6 or MRL MSCs into mouse knees immediately after fracture, and MSCs of either strain were sufficient to prevent PTA.

Genetically reprogramming adult cells into induced pluripotent stem cells (iPSCs) generates large numbers of patient-matched cells with chondrogenic potential for therapy and cartilage modeling. We produced murine iPSC-derived cartilage constructs with a multi-phase approach involving micromass culture with bone morphogenetic protein-4, flow cytometry cell sorting of chondrocyte-like cells, monolayer expansion, and pellet culture with transforming growth factor-beta 3. Successful differentiation was confirmed by increased chondrogenic gene expression, robust synthesis of glycosaminoglycans and type II collagen, and the repair of an in vitro cartilage defect.

The diverse applications pursued in this research illustrate the power of stem cells to deepen the understanding of cartilage and guide the development of therapies to prevent and treat cartilage injury and OA.