Regulation of Mesenchymal Stem Cell Multipoteniality: Interplay of Matrix Stiffness and Matrix Ligand Presentation
Mesenchymal stem cells from a variety of sources have shown enormous promise in preclinical studies to facilitate bone and cartilage regeneration. Despite this promise, the therapeutic potential of these cells relies upon identifying the necessary cues governing their self-renewal and multi-lineage maintenance. A myriad of factors are known to influence stem cell fate, including soluble and insoluble factors, cell-extracellular matrix (ECM) interactions, and mechanical forces. We hypothesize that matrix compliance and composition modulate the maintenance of mesenchymal stem cells (MSCs) in an undifferentiated state in vitro. The long-term goal is to use these cells in cartilage regeneration strategies.
Clinical application of cartilage tissue engineering strategies using adult stem cells requires constant stabilization of cell behavior, which, to date, investigators have had variable success with through ubiquitous exposure of the cells to growth factors. However, stem cells go through several stages of maturation as they become cartilage-producing cells (chondrocytes) and at each stage, the response of the cell to growth factors may be different. The objective of this proposal is to use gene transfer to concurrently manipulate pivotal intracellular pathways that regulate cartilage cell maturation and matrix remodeling as a means to direct adult stem cell maturation and matrix accumulation in vitro for development of clinically viable cartilage repair strategies.
Normal craniofacial development requires a combination of environmental and biochemical cues to promote chondrogenic differentiation and control chondrocyte maturation of ectodermal-derived neural crest cells (NCCs), which eventually give rise to craniofacial tissues. Key regulators of this process include the bone morphogenetic proteins (BMPs). Mutations in the type I BMP receptor, ALK2 (Acvr1), in mesenchymal progenitor cells can lead to heterotopic bone formation in soft tissues, but its role in NCC chondrogenesis is largely unknown. Our goal is to determine the chondrogenic potential NCCs, understand the biochemical role of bone morphogenetic protein in NCC chondorgenesis, and examine how matrix stiffness and composition contribute to chondrogenic differentiation of NCCs.
Evaluation of a Novel Bioresorbable Polymer Scaffold for the Treatment of Traumatic Cartilage Injuries
Articular cartilage injury is a major clinical problem and current surgical and tissue engineering strategies do not fully regenerate injured cartilage. Most cartilage defects inevitably lead to osteoarthritis, a painful and debilitating disease. Three dimensional polymer printing enables investigators to rapidly produce objects of complex structure in a very short time. With this technology, it would be possible to produce a scaffold that fits the geometry of a defect based on arthroscopic or magnetic resonance images of the injured area. The objective of this project is to evaluate a scaffold with a novel design whose mechanical properties will be manipulated throughout the height of the scaffold using 3D printing. We will construct a finite element model based on the specific geometries of human bones and cartilage defects using patient scans and assess the stresses produced in the tissue due to interaction with the scaffold. Several parameters of the scaffolds geometry, such as the porosity, will be varied to control the mechanical properties and prevent the formation of loading environments that have been shown to be detrimental to the health of the remaining cartilage tissue. These results will be confirmed in an in vitro model of cartilage loading and the effect on cartilage biochemistry and cell viability will be quantified. Finally, scaffolds with optimized parameters will be manufactured based on the contours of cadaveric knees and the resulting contact mechanics will be compared to that of healthy joints and knees with osteochondral defects. The ultimate goal is to produce a scaffold that will restore the contact mechanics in injured human knees to that of the intact condition, thereby preventing loading conditions that will lead to osteoarthritis.
Conditions in which cartilage calcification is aberrant affect both the new born and the elderly in terms of birth defects, growth deformities and inherited abnormalities such as achondroplasia, nutritional rickets, as well as unwanted cartilage calcification in end-stage osteoarthritis and “tissue-engineered” cartilage repair. To prevent and treat these conditions it is essential to understand physiologic cartilage calcification. We are examining the differential effects of culture conditions on chondroprogenitor cell differentiation and maturation. Specifically, we are interested in the role Pi plays in modulating chondrocyte phenotype.
Cartilage calcification is a very complex process and it is generally accepted that proteoglycans, specifically aggrecan, have a critical role in its progression. Aggrecan aggregate size is a key regulator of mineral crystal size and proliferation. However, the mechanisms by which PG morphology is regulated during cartilage calcification is still unknown. The goal of this work is to understand how aggrecan is modified during physiologic cartilage calcification. To accomplish this, we will probe cultures of maturing chondrocytes for specific neoepitopes that result from the cleavage of aggrecan by MMPs and ADAMTSs (Figure below) with monoclonal antibodies.
The 5-year survival rate of patients with bone sarcomas is 50-60%. With metastases, the survival rate drops to 20%. Despite the increase of both neoadjuvant and adjuvant chemotherapy, ~35% patients develop metastases and yet the mechanisms that drive metastatic progression of bone sarcomas remain unclear. Tumors evolve within a complex microenvironment composed of multiple ECM components that serve as cues, such as soluble and insoluble ligands, variable matrix stiffness, and exogenous mechanical loading. In spite of this knowledge being firmly established, no drugs are tested and/or designed based on 3D culture systems that are representative of realistic mechanical environments present during cancer development and progression. Moreover, focusing on 2D models may be contributing to the paucity of positive trials of new drugs, thus also highlighting the urgent need for 3D culture systems that better recapitulate the in vivo tumor microenvironment. Mechanistic studies and high-throughput analysis of rational candidate anti-cancer drugs should be conducted within 3D systems where the matrix mechanics and ECM composition are decoupled to quantitatively and systematically assess the effect of each variable in the context of the other. To this end, a physiologically-relevant and uniquely novel organoid culture system that precisely exhibits the characteristics of decoupled mechanochemical and transport properties to support tumor cell growth and angiogenic signaling is used to determine the impact of both matrix composition and stiffness on the regulation of the mechanochemical pathways associated with bone sarcoma progression, with Wnt signaling being a particularly important candidate, to identify viable, previously unexplored drug targets to exploit for therapeutic intervention.