Stem Cell Mechanobiology and Differentiation
R01 EB02425 NIH/NIBIB
Dual PIs: Dawn Elliott and Robert Mauck
Title: Multi-scale biomechanics of engineered and native fibrous load-bearing tissue
Time Period: 8/1/08-3/31/18
Meniscus architecture viewed under polarized light.
Fiber-reinforced load-bearing soft tissues, including tendon, meniscus, and annulus fibrosus, have hierarchical structure and biochemical composition that enables in vivo mechanical function. These tissues are prone to degeneration and injury, with debilitating consequences, high costs, and limited therapeutic options. Although load-bearing tissues are classically described using idealized schematics showing an ordered prevailing fiber direction, these tissues are in fact highly inhomogeneous, with amorphous proteoglycan-rich structural micro-domains within an otherwise ordered collagen-rich fibrous tissue- they are not simply a schematic. The objective of this proposal is to investigate micro-level structure-function of native and engineered tissue by quantifying and modeling mechanics from the tissue to the cellular level, and evaluating the mechanistic impact of micro-level structure-function on mechanotransduction. This study will also recapitulate these natural and/or potentially diseased micro-environments in engineered tissues in order to develop controlled in vitro systems to evaluate altered mechanotransduction. Quantifying the size, mechanical inhomogeneity, and biological response among tissue micro-domains is important because this is the length scale that governs cell mechanotransduction, and therefore regulation of tissue homeostasis and disease progression. These studies will advance the field of regenerative medicine by addressing micromechanical mechanisms in tissue development, degeneration, and injury. Ultimately, this new understanding will direct therapeutic strategies for rehabilitation, repair, and replacement to promote and preserve healthy mechanotransduction in fibrous load bearing tissues.
R01 EB008722 NIH/NIBIB
Dual PIs: Jason Burdick and Robert Mauck
Title: Engineering Developmental Microenvironments: Cartilage Formation and Maturation
Time Period: 4/13/09-3/31/18
Engineered cartilage constructs (left). Focal defect in minipig cartilage imaged by microCT (right).
Articular cartilage lines the surfaces of joints and transmits the forces generated with loading. Due to limitations in the natural healing capacity of cartilage, and given the increasing incidence of osteoarthritis, there exists a growing demand for cell-based strategies for repair. Tissue engineering, and particularly those approaches based on autologous mesenchymal stem cells (MSCs), is evolving as a clinically relevant technique to promote cartilage regeneration. Yet, the formed tissue properties as well as the stability of phenotype and heterogeneous cellular response within constructs are concerns that currently limit translation of this technology. Our general approach to MSC-based cartilage repair addresses the differences observed between early rapid stages of cartilage formation and the gradual remodeling (maturation) that results in a tissue capable of adult function. This transformative process is driven by a multitude of temporal factors (chemical, mechanical, and soluble). Our progress during the ongoing grant has shown a role for matrix and cellular density, the timing of material degradation, introduction of soluble inductive factors, and mechanical loading (both compression and sliding contact) in guiding cartilage formation and maturation. Here, we build from these studies by introducing a developmentally relevant signal, namely cell-cell interactions through N-cadherin that are found during limb bud development, into our engineered hydrogel systems. In the first Aim, MSCs will be encapsulated in HA hydrogels modified with peptides that mimic the extracellular domain of N-cadherin, and the influence of peptide density on chondrogenesis and cartilage maturation will be investigated, in addition to the influence of the peptide on population heterogeneity and phenotypic stability. In the second Aim, the temporal presentation of the peptides will be investigated by introducing linkers that undergo cell-mediated proteolysis of the peptides from the HA hydrogels. The influence of the temporal peptide presentation on chondrogenesis, cartilage maturation, population heterogeneity, and phenotypic stability will be assessed as in the first Aim, in addition to the responsiveness to mechanical loading based on the acceleration of chondrogenesis. In the third Aim, N-cadherin peptide modified hydrogels, including both stable and transient presentation, will be investigated in a clinically-relevant load-bearing porcine defect model to assess the role of these interactions in an implanted hydrogel in cartilage defect repair. These Aims were designed to allow the testing of our hypotheses that control over the MSC microenvironment, and inclusion of signals present during normal development that are both permissive and instructive for cartilage formation and maturation, will lead to the generation of constructs with properties akin to native tissue and improved repair.
R01 AR056624 NIH/NIAMS
Dual PIs: Robert Mauck and Jason Burdick
Title: Dynamic Fibrous Scaffolds for Repairing Dense Connective Tissues
Time Period: 7/1/08-8/31/19
Dynamic fibrous scaffold undergoing dissolution of green fiber fraction.
Fibrous tissues of the musculoskeletal system are plagued by their poor intrinsic healing capacity. In the previous funding cycle, we developed enabling technologies towards the production of a novel class of composite electrospun scaffolds, and used these scaffolds to develop cell-based tissue engineered constructs with native-like tissue properties and organization. In this competitive renewal, we shift our focus to using these enabling technologies to enhance endogenous tissue repair. Our focus is on the knee meniscus, a fibrous tissue critical for proper load transfer and for which current repair strategies do not restore function. The overall objective of this renewal is to use these scaffolds to deliver multiple agents over different temporal scales to specifically address the inherent limitations to endogenous meniscus repair. These limitations in the adult include synovial inflammation, low endogenous cellularity, and hindered cell mobility to the wound interface. This proposal will employ composite scaffolds (developed during the first funding cycle) that provide a stable fiber fraction (polycaprolactone (PCL), to provide an instructional pattern and mechanical stability), a sacrificial fiber fraction (polyethylene oxide (PEO), to define initial scaffold porosity), and an MMP-cleavable hyaluronic acid (HA) fiber fraction (that degrades in response to elevated proteolytic activity in synovial fluid of patients with meniscus damage). Degradation of the HA fiber fraction will both enhance cellular infiltration (by increasing scaffold porosity) and at the same time reduce degradation of nascent repair tissue (via competitive inhibition of synovial MMPs). These composite scaffolds will also address limited cellular mobility through the dense surrounding ECM by rapidly and locally decreasing nuclear stiffness (via the delivery of agents that reduce heterochromatin content and/or Lamin A/C processing) in endogenous meniscus cells. Finally, these scaffolds will selectively recruit endogenous meniscus cells towards the wound interface, to accelerate and sustain the repair process, via the delivery of stromal derived factor-1α (SDF-1α), a potent cytokine that increases meniscal cell migration. The synergistic interactions of these different repair adjuvants will be validated through in vitro scaffold and meniscal explant studies, and then tested in our large animal (ovine) meniscus defect model. If successful, these studies and technologies will set the stage for clinical translation and treatment of human meniscal injury by overcoming the inherent limitations to endogenous meniscus repair.
VA RR&D I01 RX001321 Department of Veterans Affairs Merit Award
PIs: Robert Mauck and Lachlan Smith
Title: Bioactive Injectable Implants for Functional Intervertebral Disc Regeneration
Time Period: 09/01/14-08/30/18
Normal (left) and degenerate (right) goat intervertebral disc.
Low back pain will affect up to 85 percent of people at some point during their lives, resulting in healthcare and related costs to the United States economy in excess of $100 billion every year. Among veterans, chronic low back pain accounts for over 70 percent of chiropractic visits and in many cases is directly connected to their period of service. Intervertebral disc degeneration, a progressive, inflammation driven cascade that leads to structural and mechanical failure, is strongly implicated as a cause of low back pain. Between 2001 and 2010, more than 130,000 active service members received diagnoses of disc degeneration, with annual incidence rates more than doubling over this period. Developing new treatment strategies for disc degeneration is therefore highly relevant to both active military personnel and veterans. A key limitation of current treatments for disc degeneration is that they do not seek to maintain or restore native tissue structure and mechanical function. There is therefore a strong need for new therapies for disc degeneration that retain and/or restore disc structure and mechanical function by directly addressing the underlying causes and mechanisms. The ideal therapy for disc degeneration would: 1) be minimally invasive; 2) restore biomechanical function; 3) attenuate localized inflammation driving tissue catabolism; and 4) potentiate long term extracellular matrix regeneration. We have recently developed a novel injectable hydrogel that polymerizes rapidly in the absence of exogenous cross-linking agents, supports mesenchymal stem cell survival and biosynthesis, and normalizes disc mechanical function. We have also developed a novel sustained release therapy for disc inflammation using interleukin-1 receptor antagonist (IL-1ra) delivered from polymeric microspheres. The objective of this proposal is to synergize these technologies to develop a minimally invasive therapy that simultaneously normalizes disc mechanical function, attenuates localized inflammation, and promotes stem-cell driven native tissue regeneration. We hypothesize that only by addressing these criteria will a therapy for disc degeneration have long-term efficacy. In Aim 1 we will establish in vitro techniques for preconditioning stem cells to survive in the harsh in vivo microenvironment of the disc, verify the efficacy of preconditioning in vivo, and establish the optimum in vivo seeding density which balances maximum regeneration potential with the limited nutritional availability inherent to the implantation environment. In Aim 2 we will extend our previous work to develop and test an anti-inflammatory therapy that attenuates the complex inflammatory cytokine expression profile present in the degenerate disc, with the dual objectives of preserving stem cell regenerative potential and halting continued native tissue destruction. Finally, in Aim 3, as a critical pre-clinical step we will evaluate this therapeutic strategy in an established large animal model of disc degeneration.
VA RR&D I21 RX001765 Department of Veterans Affairs SPiRE Award
PIs: Robert Mauck and Maurizio Pacifici
Title: Cartilage Repair with Synovial Joint Precursors
Time Period: 12/01/14-11/30/16
A) MicroCT without and with contrast agent of an E42 Yucatan minipig embryo. B) Individual microCT slice of forelimb and C) hindlimb.
Cartilage covers the joint surface and absorbs and transmits mechanical forces during normal activity. Given its poor healing capacity and the high incidence of cartilage disease including osteoarthritis, there exists a growing and urgent demand for cell-based repair strategies. Our group has made considerable progress in the creation of 3D synthetic microenvironments in which cells can deposit cartilage extracellular matrix (ECM) and mature into functional engineered cartilage. These studies have employed adult bone marrow derived mesenchymal stem cells (MSCs) coupled with hydrogels based on cartilage ECM (hyaluronic acid (HA)). Despite our progress in this area, several impediments remain in the clinical use of these constructs. These include the finding that MSCs fail to produce engineered tissues that match native properties, fail to fully adopt the chondrocyte phenotype, and are furthermore susceptible to unwanted phenotypic transitions in vivo (i.e., progress to a bone-like phenotype). However, recent studies by our team have identified a novel cohort of progenitor cells that are responsible for synovial joint formation in the embryo, are distinct from progenitors that form bone, and persist within articular cartilage as a progenitor pool. We hypothesize that these synovial joint-forming progenitor cells have a superior and unique capacity to generate cartilage and can be used to engineer and regenerate cartilage with native joint-associated properties. To test this hypothesis, we will develop new methods for cell isolation and expansion, will evaluate differentiation and stability of this cell population (relative to MSCs) in 3D culture, and will test their translational efficacy in forming functional cartilage in vivo in a large animal defect model. In this application, we explore the translational potential of synovial joint progenitor cells through two Aims. If successful, this work will develop a unique synovial progenitor cell source and validate the efficacy of this cell type in long-term translational studies. This work has the potential to alter clinical practice by improving patient outcomes after cartilage repair surgeries.
R01 AR041916-15S1 NIAMS Building Interdisciplinary Research Teams (BIRT) Supplement
Multi-PIs: Fred Kaplan, Eileen Shore, and Robert Mauck
Title: The Cellular and molecular Basis of FOP Lesions
Time Period: 07/04/13-03/31/16
Histological appearance and indentation mechanics of normal and fibroproliferative muscle 4 days after cardiotoxin (CTX) injury in wild type and FOP mice.
Rare genetic disorders, although directly impacting relatively small segments of the population, are caused by mutations in genes with such critical importance that changes in their function are rarely tolerated, providing unique insight into fundamental cellular mechanisms. One such disease, fibrodysplasia ossificans progressiva (FOP) is caused by misregulated control of cell fate decisions that lead to congenital skeletal malformations and progressive disabling extra-skeletal (heterotopic) endochondral ossification. We determined that all familial and sporadic cases of classic FOP carry the same heterozygous mutation in ACVR1/ALK2 (R206H; c.617G>A), a cell surface receptor that mediates signal transduction of bone morphogenetic proteins (BMPs). Our data show that ACVR1 R206H activates the BMP pathway, at least in part, through mildly activating BMP-independent signaling. Commitment and differentiation of progenitor cells are regulated by signals from the tissue microenvironment that direct cell fate to specific lineages, including BMPs that are established regulators of early development and cell differentiation. However, cells exist in vivo in a mechanical environment, experiencing local microenvironments of varying elasticity/stiffness and dynamic mechanical signals (such as tensile deformation) through physiologic activities. These mechanical signals can also direct cell fate decisions, and are mediated through some of the same pathways that transmit signals from classical soluble factors/cytokines. We propose that the R206H ACVR1 receptor mutation enhances progenitor cells to be more responsive to interactions with molecular and mechanical modulators of cell differentiation, and that in patients this enhanced sensitivity can trigger and/or mediate active episodes of endochondral bone formation. We hypothesize that enhanced BMP signaling by the ACVR1 R206H mutation alters the normal cell differentiation "set-point" of mesenchymal stem cells, increasing the sensitivity of these cells to microenvironmental mechanical cues that modulate cell fate decisions. A new multi-disciplinary team of investigators will work together on this BIRT proposal to accomplish two specific aims. Aim 1: To investigate the chondrogenic response of Acvr1R206H mutant cells to static mechanical forces and altered cell mechanics in the cell microenvironment. This Aim will examine differences in the internal cellular contractile machinery in cells with and without the Acvr1R206H mutation, and their response to changes in the elasticity (substrate stiffness) of the niche. Aim 2: To investigate the chondrogenic response of Acvr1R206H mutant cells to active mechanical forces (cell deformation) from the cell microenvironment. This Aim will examine the interactions of the ACVR1 R206H mutation with externally applied mechanical forces that alter cell shape (tensile deformation of the niche). The proposed highly innovative investigations will be conducted by a new and synergistic, multi-disciplinary, and interactive research team in order to identify regulatory mechanisms controlling cell differentiation and provide the foundation for establishing a new and innovative multidisciplinary research program.
URF Research Award – University Research Foundation
PIs: Robert Mauck and Vivek Shenoy
Title: Mapping and Modeling Mechanical Force Transduction in Mesenchymal Stem Cells
Time Period: 03/01/14-08/31/15
Actin (green), DAPI (blue), and calmodulin (red) in a naïve mesenchymal stem cell (left). Mesenchymal stem cell on a stiff substrate (right).
Differentiation of mesenchymal stem cells (MSCs) directs cell function and can be mediated by soluble and biophysical cues. Recent evidence suggests that differentiation also alters cellular mechanics. Our data show that differentiation stiffens MSC nuclei as the Lamin A/C network reorganizes and heterochromatin increases. These changes increase nuclear stiffness and sensitize cells to mechanical perturbation. Additionally, we have shown that both the cytoskeletal contractility and its nuclear connectivity, which both change with differentiation, are important in determining response to mechanical signals. These data suggest that multiple biophysical properties of stem cells change with differentiation, and that these changes alter the manner in which these cells respond to their mechanical environment. In the proposed work, Dr. Mauck (Orthopaedic Surgery and Bioengineering) and Dr. Shenoy (Materials Science) will build a new collaboration to investigate the manner in which force transduction occurs from the extracellular environment to the nucleus, and how this impacts stem cell fate determination and response to mechanical cues. In doing so, we will construct sophisticated models of cellular machinery mediating force transduction, and perturb each model element through experimental manipulation of nuclear stiffness, nuclear connectivity, cytoskeletal contractility, and extracellular mechanics and deformation.