development-of-a-uterosacral-ligament-suspension-rat-model

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

University of Virginia

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.

Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates.

Promoting remyelination and/or minimizing demyelination are key therapeutic strategies under investigation for diseases and injuries like multiple sclerosis (MS), spinal cord injury, stroke, and virus-induced encephalopathy. Myelination is essential for efficacious neuronal signaling. This myelination process is originated by oligodendrocyte progenitor cells (OPCs) in the central nervous system (CNS). Resident OPCs are capable of both proliferation and differentiation, and also migration to demyelinated injury sites. OPCs can then engage with these unmyelinated or demyelinated axons and differentiate into myelin-forming oligodendrocytes (OLs). However this process is frequently incomplete and often does not occur at all. Biomaterial strategies can now be used to guide OPC and OL development with the goal of regenerating healthy myelin sheaths in formerly damaged CNS tissue. Growth and neurotrophic factors delivered from such materials can promote proliferation of OPCs or differentiation into OLs. While cell transplantation techniques have been used to replace damaged cells in wound sites, they have also resulted in poor transplant cell viability, uncontrollable differentiation, and poor integration into the host. Biomaterial scaffolds made from extracellular matrix (ECM) mimics that are naturally or synthetically derived can improve transplanted cell survival, support both transplanted and endogenous cell populations, and direct their fate. In particular, stiffness and degradability of these scaffolds are two parameters that can influence the fate of OPCs and OLs. The future outlook for biomaterials research includes 3D in vitro models of myelination / remyelination / demyelination to better mimic and study these processes. These models should provide simple relationships of myelination to microenvironmental biophysical and biochemical properties to inform improved therapeutic approaches.

Engineering biomaterial microenvironments to promote myelination in the central nervous system.

Viscoelasticity has emerged as a critical regulator of cell behavior. However, there is an unmet need to develop biomaterials where viscoelasticity can be spatiotemporally controlled to mimic the dynamic and heterogeneous nature of tissue microenvironments. Toward this objective, we developed a modular hyaluronic acid hydrogel combining light-mediated covalent and supramolecular cross-linking to afford spatiotemporal control of network viscoelastic properties. Covalently cross-linked elastic hydrogels or viscoelastic hydrogels combining covalent and supramolecular interactions were fabricated to match healthy and fibrotic liver mechanics. LX-2 human hepatic stellate cells cultured on viscoelastic hydrogels displayed reductions in spreading, actin stress fiber organization, and myocardin-related transcription factor A (MRTF-A) nuclear localization compared to cells on elastic hydrogels. We further demonstrated the dynamic capabilities of our hydrogel system through photo-mediated secondary incorporation of either covalent or supramolecular cross-links to modulate viscoelastic properties. We used photopatterning to create hydrogels with well-controlled patterned regions of stiff elastic mechanics representing fibrotic tissue nodules surrounded by regions of soft viscoelastic hydrogel mimicking healthy tissue. Cells responded to the local mechanics of the patterned substrates with increased spreading in fibrosis-mimicking regions. Together, this work represents an important step forward toward the creation of hydrogel models with spatiotemporal control of both stiffness and viscoelastic cell-instructive cues.

Spatiotemporal Control of Viscoelasticity in Phototunable Hyaluronic Acid Hydrogels.

The lack of regenerative solutions for demyelination within the central nervous system motivates the development of strategies to expand and drive the bioactivity of the cells, including oligodendrocyte progenitor cells (OPCs), that ultimately give rise to myelination. In this work, we introduce a 3D hyaluronic acid (HA) hydrogel system to study the effects of microenvironmental mechanical properties on the behavior of OPCs. We tuned the stiffness of the hydrogels to match the brain tissue (storage modulus 200-2000 Pa) and studied the effects of stiffness on metabolic activity, proliferation, and cell morphology of OPCs over a 7 day period. Although hydrogel mesh size decreased with increasing stiffness, all hydrogel groups facilitated OPC proliferation and mitochondrial metabolic activity to similar degrees. However, OPCs in the two lower stiffness hydrogel groups (170 ± 42 and 794 ± 203 Pa) supported greater adenosine triphosphate levels per cell than the highest stiffness hydrogels (2179 ± 127 Pa). Lower stiffness hydrogels also supported higher levels of cell viability and larger cell spheroid formation compared to the highest stiffness hydrogels. Together, these data suggest that 3D HA hydrogels are a useful platform for studying OPC behavior and that OPC growth/metabolic health may be favored in lower stiffness microenvironments mimicking brain tissue mechanics.

3D Hyaluronic Acid Hydrogels for Modeling Oligodendrocyte Progenitor Cell Behavior as a Function of Matrix Stiffness.

There is often a tradeoff between in vitro disease modeling platforms that capture pathophysiologic complexity and those that are amenable to high-throughput fabrication and analysis. However, this divide is closing through the application of a handful of fabrication approaches-parallel fabrication, automation, and flow-driven assembly-to design sophisticated cellular and biomaterial systems. The purpose of this review is to highlight methods for the fabrication of high-throughput biomaterial-based platforms and showcase examples that demonstrate their utility over a range of throughput and complexity. We conclude with a discussion of future considerations for the continued development of higher-throughput in vitro platforms that capture the appropriate level of biological complexity for the desired application. STATEMENT OF SIGNIFICANCE: There is a pressing need for new biomedical tools to study and understand disease. These platforms should mimic the complex properties of the body while also permitting investigation of many combinations of cells, extracellular cues, and/or therapeutics in high-throughput. This review summarizes emerging strategies to fabricate biomimetic disease models that bridge the gap between complex tissue-mimicking microenvironments and high-throughput screens for personalized medicine.

Fabrication approaches for high-throughput and biomimetic disease modeling.

The fibrous architecture of the extracellular matrix (ECM) is recognized as an integral regulator of cell function. However, there is an unmet need to develop mechanically robust biomaterials mimicking nanofibrous tissue topography that are also injectable to enable minimally invasive delivery. In this study, we have developed a fibrous hydrogel composed of supramolecularly assembled hyaluronic acid (HA) nanofibers that exhibits mechanical integrity, shear-thinning behavior, rapid self-healing, and cytocompatibility. HA was modified with methacrylates to permit fiber photo-cross-linking following electrospinning and either "guest" adamantane or "host" β-cyclodextrin groups to guide supramolecular fibrous hydrogel assembly. Analysis of fibrous hydrogel rheological properties showed that the mixed guest-host fibrous hydrogel was more mechanically robust (6.6 ± 2.0 kPa, storage modulus (')) than unmixed guest hydrogel fibers (1.0 ± 0.1 kPa) or host hydrogel fibers (1.1 ± 0.1 kPa) separately. The reversible nature of the guest-host supramolecular interactions also allowed for shear-thinning and self-healing behavior as demonstrated by cyclic deformation testing. Human mesenchymal stromal cells (hMSCs) encapsulated in fibrous hydrogels demonstrated satisfactory viability following injection and after 7 days of culture (>85%). Encapsulated hMSCs were more spread and elongated when cultured in viscoelastic guest-host hydrogels compared to nonfibrous elastic controls, with hMSCs also showing significantly decreased circularity in fibrous guest-host hydrogels compared to nonfibrous guest-host hydrogels. Together, these data highlight the potential of this injectable fibrous hydrogel platform for cell and tissue engineering applications requiring minimally invasive delivery.

Guest-Host Supramolecular Assembly of Injectable Hydrogel Nanofibers for Cell Encapsulation.

Skeletal muscle is characterized by its three-dimensional (3D) anisotropic architecture composed of highly aligned and electrically-excitable muscle fibers that enable normal movement. Biomaterial-based tissue engineering approaches to repair skeletal muscle are limited due to difficulties combining 3D structural alignment (to guide cell/matrix organization) and electrical conductivity (to enable electrically-excitable myotube assembly and maturation). In this work we successfully produced aligned and electrically conductive 3D collagen scaffolds using a freeze-drying approach. Conductive polypyrrole (PPy) nanoparticles were synthesized and directly mixed into a suspension of type I collagen and chondroitin sulfate followed by directional lyophilization. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and confocal microscopy showed that directional solidification resulted in scaffolds with longitudinally aligned pores with homogeneously-distributed PPy content. Chronopotentiometry verified that PPy incorporation resulted in a five-fold increase in conductivity compared to non-PPy-containing collagen scaffolds without detrimentally affecting myoblast metabolic activity. Furthermore, the aligned scaffold microstructure provided contact guidance cues that directed myoblast growth and organization. Incorporation of PPy also promoted enhanced myotube formation and maturation as measured by myosin heavy chain (MHC) expression and number of nuclei per myotube. Together these data suggest that aligned and electrically conductive 3D collagen scaffolds could be useful for skeletal muscle tissue engineering.

Aligned and electrically conductive 3D collagen scaffolds for skeletal muscle tissue engineering.

Volumetric muscle loss (VML) injuries are characterized by permanent loss of muscle mass, structure, and function. Hydrogel biomaterials provide an attractive platform for skeletal muscle tissue engineering due to the ability to easily modulate their biophysical and biochemical properties to match a range of tissue characteristics. In this work, we successfully developed a mechanically tunable hyaluronic acid (HA) hydrogel system to investigate the influence of hydrogel stiffness on VML repair. HA was functionalized with photoreactive norbornene groups to create hydrogel networks that rapidly crosslink through thiol-ene click chemistry with tailored mechanics. Mechanical properties were controlled by modulating the amount of matrix metalloproteinase-degradable peptide crosslinker to produce hydrogels with increasing elastic moduli of 1.1 ± 0.002, 3.0 ± 0.002, and 10.6 ± 0.006 kPa, mimicking a relevant range of developing and mature muscle stiffnesses. Functional muscle recovery was assessed following implantation of the HA hydrogels by  photopolymerization into rat latissimus dorsi (LD) VML defects at 12 and 24 weeks postinjury. After 12 weeks, muscles treated with medium stiffness (3.0 kPa) hydrogels produced maximum isometric forces most similar to contralateral healthy LD muscles. This trend persisted at 24 weeks postinjury, suggestive of sustained functional recovery. Histological analysis revealed a significantly larger zone of regeneration with more  muscle fibers following implantation of medium stiffness hydrogels in VML-injured muscles compared to other experimental groups. Lower (low and medium) stiffness hydrogels also appeared to attenuate the chronic inflammatory response characteristic of VML injuries, displaying similar levels of macrophage infiltration and polarization to healthy muscle. Together these findings illustrate the importance of hydrogel mechanical properties in supporting functional repair of VML injuries. Impact statement This report defines the role hydrogel mechanical properties play in the repair of volumetric muscle loss (VML) injuries. We show that an intermediate hydrogel stiffness (3 kPa) more compliant than adult muscle tissue facilitated improved and sustained regenerative outcomes up to 24 weeks postinjury in a rat latissimus dorsi model of VML. Muscles treated with 3 kPa hydrogels showed enhanced myogenesis and attenuation of the chronic inflammatory response characteristic of VML injuries. These results should help guide the future design of hydrogels for skeletal muscle tissue engineering and regeneration.

Photoreactive Hydrogel Stiffness Influences Volumetric Muscle Loss Repair.

Tissue fibrosis is characterized by progressive extracellular matrix (ECM) stiffening and loss of viscoelasticity that ultimately impairs organ functionality. Cells bind to the ECM through integrins, where  integrin engagement in particular has been correlated with fibroblast activation into contractile myofibroblasts that drive fibrosis progression. There is a significant unmet need for  hydrogel systems that deconstruct the complexity of native tissues to better understand the individual and combined effects of stiffness, viscoelasticity, and integrin engagement on fibroblast behavior.

The Combined Influence of Viscoelastic and Adhesive Cues on Fibroblast Spreading and Focal Adhesion Organization.

Skeletal muscle's combination of three-dimensional (3D) anisotropy and electrical excitability is critical for enabling normal movement. We previously developed a 3D aligned collagen scaffold incorporating conductive polypyrrole (PPy) particles to recapitulate these key muscle properties and showed that the scaffold facilitated enhanced myotube maturation compared with nonconductive controls. To further optimize this scaffold design, this work assessed the influence of conductive polymer incorporation and scaffold pore architecture on myogenic cell behavior. Conductive PPy and poly(3,4-ethylenedioxythiophene) (PEDOT) particles were synthesized and mixed into a suspension of type I collagen and chondroitin sulfate prior to directional freeze-drying to produce anisotropic scaffolds. Energy dispersive spectroscopy revealed homogenous distribution of conductive PEDOT particles throughout the scaffolds that resulted in a threefold increase in electrical conductivity while supporting similar myoblast metabolic activity compared to nonconductive scaffolds. Control of freezing temperature enabled fabrication of PEDOT-doped scaffolds with a range of pore diameters from 98 to 238 μm. Myoblasts conformed to the anisotropic contact guidance cues independent of pore size to display longitudinal cytoskeletal alignment. The increased specific surface area of the smaller pore scaffolds helped rescue the initial decrease in myoblast metabolic activity observed in larger pore conductive scaffolds while also promoting modestly increased expression levels of the myogenic marker myosin heavy chain (MHC) and gene expression of myoblast determination protein (MyoD). However, cell infiltration to the center of the scaffolds was marginally reduced compared with larger pore variants. Together these data underscore the potential of aligned and PEDOT-doped collagen scaffolds for promoting myogenic cell organization and differentiation.

Article	Year
Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29507238	2018
Engineering biomaterial microenvironments to promote myelination in the central nervous system. Brain research bulletin\| PubMed ID: 31306690	2019
Spatiotemporal Control of Viscoelasticity in Phototunable Hyaluronic Acid Hydrogels. Biomacromolecules\| PubMed ID: 31600072	2019
3D Hyaluronic Acid Hydrogels for Modeling Oligodendrocyte Progenitor Cell Behavior as a Function of Matrix Stiffness. Biomacromolecules\| PubMed ID: 33112592	2020
Fabrication approaches for high-throughput and biomimetic disease modeling. Acta biomaterialia\| PubMed ID: 33716174	2021
Guest-Host Supramolecular Assembly of Injectable Hydrogel Nanofibers for Cell Encapsulation. ACS biomaterials science & engineering\| PubMed ID: 33891397	2021
Aligned and electrically conductive 3D collagen scaffolds for skeletal muscle tissue engineering. Biomaterials science\| PubMed ID: 33899845	2021
Photoreactive Hydrogel Stiffness Influences Volumetric Muscle Loss Repair. Tissue engineering. Part A\| PubMed ID: 34409861	2022
The Combined Influence of Viscoelastic and Adhesive Cues on Fibroblast Spreading and Focal Adhesion Organization. Cellular and molecular bioengineering\| PubMed ID: 34777602	2021
Controlling scaffold conductivity and pore size to direct myogenic cell alignment and differentiation. Journal of biomedical materials research. Part A\| PubMed ID: 35762455	2022

Steven R. Caliari

Biography

JoVE Journal Publications

Other Publications