Translational Models of Tissue Repair and Regeneration
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.
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 I01 RX001213 Department of Veterans Affairs Merit Award
PIs: George Dodge and Robert Mauck
Title: Cartilage response to compression injury: A platform for therapeutics discovery
Time Period: 09/01/14-08/30/18
High throughput mechanical injury of engineered cartilage.
This project seeks to rapidly advance the state of the art in understanding and discovering new therapeutic agents for the treatment of traumatic injury to cartilage. Excessive loads and blunt force trauma are responsible for initiating and furthering cartilage pathology and eventually an "under repaired" joint surface that progressively deteriorates and functionally fails. Indeed, patients with focal cartilage defects have quality of life scores comparable to those with severe osteoarthritis (OA), further emphasizing the need for early intervention. Post-traumatic OA (PTOA) defines the subset of OA patients whose cartilage pathology emerged directly as a consequence of trauma to the joint and in fact probably started with the traumatic injury to the joint. PTOA is widespread in both the general and military population, and is largely untreatable and untreated in current clinical practice. The challenge in cartilage repair after such injuries is the inherent poor healing capacity of the native tissue and the lack of molecules that can be used at the time of injury to preserve cell viability, biosynthetic activities, and foster intrinsic repair. Moreover, there is currently no high throughput screening method that models this injury state, and so no clear route forward for the rapid identification of novel therapeutics that could improve clinical practice and patient outcomes after injury. Our design platform is centered on the development of cartilage-like tissues on the micro-scale and in large quantities. We then mechanically impact these cartilage tissue analogs (CTAs) and assess their cellular and overall degenerative response as a function of time subsequent to insult. In Aim 1 of this proposal, we will scale up an existing validated high throughput testing system to accommodate even larger sample numbers and develop rapid and cost effective outcome measures specific to degradative signaling in cartilage after injury, thus making the testing platform suitable for high throughput screening (HTS). In Aim 2, we will validate this novel device in conjunction with engineered CTAs and native tissue, so as to match the timing and magnitude of key signaling events that occur after injury. In Aim 3, we will use this validated system to screen commercially available small molecule libraries in order to identify molecules important in chondrocyte response to injury. In Aim 4, we will use both soluble and biomaterial-mediated delivery systems in order to test the therapeutic efficacy of identified compounds (and their combinations) in human tissue analogs and native human cartilage response to injury. These delivery systems are designed to enable rapid translation to subsequent pre-clinical animal models and ultimately to human clinical trials. The study is highly translational in that it relates to the all too common instance of blunt force trauma and injury to cartilage, a condition extremely prevalent in the active duty military population, and will provide a novel and much needed testing platform for small molecule discovery in this clinical domain.
AO Foundation Acute Cartilage Injury (ACI) Consortium Grant
PIs: Robert Mauck and George Dodge
Title: A novel platform for optimizing material design for cartilage tissue engineering
Time Period: 04/01/11-12/31/16
Implanted woven PCL scaffold in a minipig focal cartilage defect.
The ultimate aim of this international ACI consortium is to develop collaborative methods to improve the repair of acute chondral defects. Within the consortium a large animal (minipig), trochlear groove, full thickness chondral defect model has been developed. During the current funding cycle, the model will now be used to investigate a number of material / adeno associated virus (AAV) conditions at two sites; Homburg and Philadelphia. Distribution of the materials across two sites ensures robustness of the results obtained. Implanted constructs will be evaluated at a 6 month time point, with implants subjected to extensive testing (µCT, MRI, mechanical testing and histological evaluation) in order to establish their efficacy in functional cartilage repair. In addition to carrying out implant testing in the large animal model, Drs. Mauck and Dodge will lead mechanical and histological evaluation of implants at the Philadelphia site.
VA RR&D I01 RX000700 Department of Veterans Affairs Merit Award
PI: David Steinberg
Title: Cartilage Repair in a Large Animal Model with Stem Cell Based Hydrogel Constructs
Time Period: 07/01/12-06/30/16
Surgical model of focal cartilage defects in a Yucatan minipig (left). Integration of repair construct in focal cartilage defect(right).
Intrinsic repair of articular cartilage damage is unsatisfactory, largely due to its avascular nature and demanding physical environment. Hydrogels are attractive biomaterials for cartilage regeneration, as they can conform within complex chondral defects, adhere to and integrate with surrounding tissues, and encapsulate and direct stem cell differentiation. The aim of this study is to evaluate a novel hyaluronic acid hydrogel seeded with mesenchymal stem cells in a cartilage defect in a large animal model. We will specifically address two different approaches for cartilage restoration using this system: 1) direct fabrication of the engineered material in the cartilage defect, where differentiation and maturation is controlled by co-encapsulated growth factor-laden microspheres, and 2) implantation of an engineered construct that has been pre-matured ex vivo under defined conditions to attain cartilage-relevant mechanical and biochemical properties. Optimization of each approach will involve detailed analysis of cartilage properties after implantation using advanced mechanical and imaging modalities. If successful, this work has the potential to dramatically change the course of treatment of persons with significant cartilage injuries and osteoarthritic degeneration, and as such, would improve the lives of military personnel and society as a whole.
VA RR&D I01 RX000979 Department of Veterans Affairs Merit Award
PI: Joe Bernstein
Title: The role of Local NSAID Administration and Inflammation on Tendon Healing
Time Period: 10/01/13-9/30/17
Restoration of function following tendon injuries stemming from military trauma, injury, overuse, or degenerative disease is largely dependent on the reestablishment of the muscle-tendon-bone connection with minimal scarring between the tendon and its surrounding tissues. Current clinical treatment following tendon repairs often includes prescribing non-steroidal anti-inflammatory drugs (NSAIDs), chiefly for their painrelieving effects, but potentially for limiting inflammation as well. By limiting pain, NSAIDs facilitate rehabilitation. In addition, NSAIDs may reduce the cross-sectional area of a tendon repair- a feature beneficial in clinical situations where thickening of a healing tendon is a problem - e.g., in the hand or shoulder. Conversely, NSAIDs can inhibit tendon cell migration and proliferation and decrease the strength of repaired tendon injuries. The correct amount of NSAID administration, as well as its timing, is not known. As such, the objective of this study is to investigate the effects, dosage requirements, and kinetics of NSAID use in the early phases of tendon repair. The study design employs novel nanofibrous biomaterial scaffolds and a microsphere drug delivery system for tendon regeneration. A well-established rat rotator cuff supraspinatus tendon injury and repair model will be used to evaluate the efficacy of the engineered constructs in vivo.
VA RR&D I01 RX000174 Department of Veterans Affairs Merit Award
Multi-PIs: Robert Mauck and John Esterhai
Title: Engineered Multi-Functional Nanofibrous Meniscus Implants
Time Period: 02/01/10-12/31/18
Scaffold mediated repair of native meniscus.
Fibrous tissues of the musculoskeletal system are plagued by their poor intrinsic healing capacity. In this proposal, we focus on the knee meniscus, a tissue critical for proper load transfer, and for which current repair strategies do not restore function. To address this clinical need, we have devised a novel strategy employing anisotropic biodegradable composite nanofibrous scaffolds that serve as an inter-positional device and enhance meniscus repair. The objective of this study is to develop nanofibrous scaffolds capable of releasing multiple agents over different temporal scales, and thus, positively impact the entire healing process. This proposal will utilize a novel class of composite nanofibrous scaffolds (developed during the first funding cycle) that have varying degradation profiles and release kinetics of bioactive agents targeted to different phases of healing and repair. These release profiles will be tuned to first 'enable' repair by rapidly and locally degrading the dense extracellular matrix at the injury site to allow cell migration and new tissue formation. Second, these scaffolds will selectively recruit progenitor cells to the wound interface, to accelerate and sustain the repair process. Finally, the scaffold will provide biochemical factors over a longer-term to 'direct' cell phenotype and promote matrix production. The synergistic interactions of these different adjuvants to repair will be investigated in a large animal meniscus defect model. If successful, these studies and new technologies will set the stage for clinical translation and treatment of human meniscal pathology. These novel scaffolds can be directly utilized in clinical procedures to enhance meniscus repair. The proposed research is highly relevant to the mission of the Veterans Health Administration since it will advance the health of veterans suffering from meniscus injuries resulting from military trauma, injury or from degenerative diseases. If successful, the proposed therapy will improve the quality of life of such individuals.
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 (5mm scale). 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.
VA RR&D IK2 RX001476-01 Department of Veterans Affairs Career Development Award
PI: Harvey Smith
Title: Tissue-engineered Constructs for Treatment of Intervertebral Disc Degeneration
Time Period: 10/01/14-9/31/19
Engineered disc composed of stem cell seeded HA and PCL components.
The objective of this project is to develop a tissue-engineered construct including an engineered nucleus pulposus and annulus fibrosus to treat defects of the annulus fibrosus (AF) and degenerative changes of the intervertebral disc (IVD). Three Specific Aims are pursued. Specific Aim 1 will consist of establishing in vitro tissue engineered NP fabrication from rabbit and human NP and MSC cells in hyaluronic acid (HA)-based hydrogel constructs, and evaluation of cell viability, gene regulation, ECM production, and biomechanical properties as a function of gel biophysical properties, cell seeding density, and extended culture in a disc-like environment. Specific Aim 2 will entail establishing and evaluating cellular infiltration and functional maturation engineered AF-like structures starting from rabbit and human AF and MSC seeded nanofibrous scaffolds in vitro. Specific Aim 3 will combine the tissue-engineered NP and AF developed in Aims 1 and 2 to form a composite disc-like construct, and to then apply this construct to an in vivo rabbit disc model of total discectomy. Efficacy will be assessed via MRI T2 mapping and subsequent ex vivo analysis of motion segment mechanical, biochemical, and histologic properties. If successful, this study will offer an increased understanding of degenerative disc disease and potential novel therapeutic treatment to both reverse degenerative changes as well as decrease risk of recurrent disc herniation. Further, it will provide critical support for Dr. Smith as he develops a research-intensive clinical practice in spine surgery at the Philadelphia VA Medical Center and the University of Pennsylvania.