A Rheological Study on the Effect of Tethering Pro- and Anti-Inflammatory Cytokines into Hydrogels on Human Mesenchymal Stem Cell Migration, Degradation, and Morphology.

Polymer-peptide hydrogels are being designed as implantable materials that deliver human mesenchymal stem cells (hMSCs) to treat wounds. Most wounds can progress through the healing process without intervention. During the normal healing process, cytokines are released from the wound to create a concentration gradient, which causes directed cell migration from the native niche to the wound site. Our work takes inspiration from this process and uniformly tethers cytokines into the scaffold to measure changes in cell-mediated degradation and motility. This is the first step in designing cytokine concentration gradients into the material to direct cell migration. We measure changes in rheological properties, encapsulated cell-mediated pericellular degradation and migration in a hydrogel scaffold with covalently tethered cytokines, either tumor necrosis factor-α (TNF-α) or transforming growth factor-ß (TGF-ß). TNF-α is expressed in early stages of wound healing causing an inflammatory response. TGF-ß is released in later stages of wound healing causing an anti-inflammatory response in the surrounding tissue. Both cytokines cause directed cell migration. We measure no statistically significant difference in modulus or the critical relaxation exponent when tethering either cytokine in the polymeric network without encapsulated hMSCs. This indicates that the scaffold structure and rheology is unchanged by the addition of tethered cytokines. Increases in hMSC motility, morphology and cell-mediated degradation are measured using a combination of multiple particle tracking microrheology (MPT) and live-cell imaging in hydrogels with tethered cytokines. We measure that tethering TNF-α into the hydrogel increases cellular remodeling on earlier days postencapsulation and tethering TGF-ß into the scaffold increases cellular remodeling on later days. We measure tethering either TGF-ß or TNF-α enhances cell stretching and, subsequently, migration. This work provides rheological characterization that can be used to design new materials that present chemical cues in the pericellular region to direct cell migration.

Osteogenic effects of covalently tethered rhBMP-2 and rhBMP-9 in an MMP-sensitive PEG hydrogel nanocomposite.

While bone morphogenic protein-2 (BMP-2) is one of the most widely studied BMPs in bone tissue engineering, BMP-9 has been purported to be a highly osteogenic BMP. This work investigates the individual osteogenic effects of recombinant human (rh) BMP-2 and rhBMP-9, when tethered into a hydrogel, on encapsulated human mesenchymal stem cells (MSCs). A matrix-metalloproteinase (MMP)-sensitive hydrogel nanocomposite, comprised of poly(ethylene glycol) crosslinked with MMP-sensitive peptides, tethered RGD, and entrapped hydroxyapatite nanoparticles was used. The rhBMPs were functionalized with free thiols and then covalently tethered into the hydrogel by a thiol-norbornene photoclick reaction. rhBMP-2 retained its full bioactivity post-thiolation, while the bioactivity of rhBMP-9 was partially reduced. Nonetheless, both rhBMPs were highly effective at enhancing osteogenesis over 12-weeks in a chemically-defined medium. Expression of ID1 and osterix, early markers of osteogenesis; collagen type I, a main component of the bone extracellular matrix (ECM); and osteopontin, bone sialoprotein II and dentin matrix protein I, mature osteoblast markers, increased with increasing concentrations of tethered rhBMP-2 or rhBMP-9. When comparing the two BMPs, rhBMP-9 led to more rapid collagen deposition and greater mineralization long-term. In summary, rhBMP-2 retained its bioactivity post-thiolation while rhBMP-9 is more susceptible to thiolation. Despite this shortcoming with rhBMP-9, both rhBMPs when tethered into this hydrogel, enhanced osteogenesis of MSCs, leading to a mature osteoblast phenotype surrounded by a mineralized ECM. STATEMENT OF SIGNIFICANCE: Osteoinductive hydrogels are a promising vehicle to deliver mesenchymal stem cells (MSCs) for bone regeneration. This study examines the in vitro osteoinductive capabilities when tethered bone morphogenic proteins (BMPs) are incorporated into a degradable biomimetic hydrogel with cell adhesive ligands, matrix metalloproteinase sensitive crosslinks for cell-mediated degradation, and hydroxyapatite nanoparticles. This study demonstrates that BMP-2 is readily thiolated and tethered without loss of bioactivity while bioactivity of BMP-9 is more susceptible to immobilization. Nonetheless, when either BMP2 or BMP9 are tethered into this hydrogel, osteogenesis of human MSCs is enhanced, bone extracellular matrix is deposited, and a mature osteoblast phenotype is achieved. This bone-biomimetic hydrogel is a promising design for stem cell-mediated bone regeneration.

Measuring the Effects of Cytokines on the Modification of Pericellular Rheology by Human Mesenchymal Stem Cells.

Implantable hydrogels are designed to treat wounds by providing structure and delivering additional cells to damaged tissue. These materials must consider how aspects of the native wound, including environmental chemical cues, affect and instruct delivered cells. One cell type researchers are interested in delivering are human mesenchymal stem cells (hMSCs) due to their importance in healing. Wound healing involves recruiting and coordinating a variety of cells to resolve a wound. hMSCs coordinate the cellular response and are signaled to the wound by cytokines, including transforming growth factor-ß (TGF-ß) and tumor necrosis factor-α (TNF-α), present in vivo. These cytokines change hMSC secretions, regulating material remodeling. TGF-ß, present from inflammation through remodeling, directs hMSCs to reorganize collagen, increasing extracellular matrix (ECM) structure. TNF-α, present primarily during inflammation, cues hMSCs to clear debris and degrade ECM. Because cytokines change how hMSCs degrade their microenvironment and are naturally present in the wound, they also affect how hMSCs migrate out of the scaffold to conduct healing. Therefore, the effects of cytokines on hMSC remodeling are important when designing materials for cell delivery. In this work, we encapsulate hMSCs in a polymer-peptide hydrogel and incubate the scaffolds in media with TGF-ß or TNF-α at concentrations similar to those in wounds. Multiple particle tracking microrheology (MPT) measures hMSC-mediated scaffold degradation in response to these cytokines, which mimics aspects of the in vivo microenvironment post-implantation. MPT uses video microscopy to measure Brownian motion of particles in a material, quantifying structure and rheology. Using MPT, we measure increased hMSC-mediated remodeling when cells are exposed to TNF-α and decreased remodeling after exposure to TGF-ß when compared to untreated hMSCs. This agrees with previous studies that measure: (1) TNF-α encourages matrix reorganization and (2) TGF-ß signals the formation of new matrix. These results enable material design that anticipates changes in remodeling after implantation, improving control over hMSC delivery and healing.

Human mesenchymal stem cell-engineered length scale dependent rheology of the pericellular region measured with bi-disperse multiple particle tracking microrheology.

Biological materials have length scale dependent structure enabling complex cell-material interactions and driving cellular processes. Synthetic biomaterials are designed to mimic aspects of these biological materials for applications including enhancing cell delivery during wound healing. To mimic native microenvironments, we must understand how cells manipulate their surroundings over several length scales. Our work characterizes length scale dependent rheology in a well-established 3D cell culture platform for human mesenchymal stem cells (hMSCs). hMSCs re-engineer their microenvironment through matrix metalloproteinase (MMP) secretions and cytoskeletal tension. Remodeling occurs across length scales: MMPs degrade cross-links on nanometer scales resulting in micrometer-sized paths that hMSCs migrate through, eventually resulting in bulk scaffold degradation. We use multiple particle tracking microrheology (MPT) and bi-disperse MPT to characterize hMSC-mediated length scale dependent pericellular remodeling. MPT measures particle Brownian motion to calculate rheological properties. We use MPT to measure larger length scales with 4.5 µm particles. Bi-disperse MPT simultaneously measures two different length scales (0.5 and 2.0 µm). We measure that hMSCs preferentially remodel larger length scales measured as a higher mobility of larger particles. We inhibit cytoskeletal tension by inhibiting myosin-II and no longer measure this difference in particle mobility. This indicates that cytoskeletal tension is the source of cell-mediated length scale dependent rheological changes. Particle mobility correlates with cell speed across length scales, relating material rheology to cell behavior. These results quantify length scale dependent pericellular remodeling and provide insight into how these microenvironments can be designed into materials to direct cell behavior.