Fund Black scientists.

Our nationwide network of BME women faculty collectively argue that racial funding disparity by the National Institutes of Health (NIH) remains the most insidious barrier to success of Black faculty in our profession. We thus refocus attention on this critical barrier and suggest solutions on how it can be dismantled.

Privileges & Pathways Through an Academic Career in Biomechanics: Appreciation for the 2022 ASME HR Lissner Medal.

Let me begin by sharing my deepest appreciation to the ASME for honoring me with the HR Lissner Medal and to the Journal of Biomechanical Engineering for this opportunity to share my personal path through biomechanics. ASME has been an academic home for me since my days as a doctoral student where my PhD advisors, Van C. Mow and W. Michael Lai, first supported my presenting on original research in the poster sessions and student competition of the Winter Annual Meetings. ASME meetings were where I met so many career advisors including Bob Nerem, Shu Chien, Savio Woo, Sheldon Weinbaum, Mort Friedman, Steve Goldstein, and Larry Taber who shared insights and tips to support me in navigating the bio-engineering discipline. Each of these mentors and advisors previously received the HR Lissner Medal and to be added to this community brings me the greatest sense of belonging. As I hope to convey here and as I did in my 2022 talk, I very much share this honor with numerous talented trainees that have led and motivated much of the directions in my own research program. For more than 30 years, I benefited from this collective of individuals who provided energy, innovation, talent and shared wisdom that brings me to where I stand now and is a testament to the importance of mentoring in the community of Lissner Medalists and ASME.

Hydraulic permeability and compressive properties of porcine and human synovium.

The synovium is a multilayer connective tissue separating the intra-articular spaces of the diarthrodial joint from the extra-synovial vascular and lymphatic supply. Synovium regulates drug transport into and out of the joint, yet its material properties remain poorly characterized. Here, we measured the compressive properties (aggregate modulus, Young's modulus, and Poisson's ratio) and hydraulic permeability of synovium with a combined experimental-computational approach. A compressive aggregate modulus and Young's modulus for the solid phase of synovium were quantified from linear regression of the equilibrium confined and unconfined compressive stress upon strain, respectively (HA = 4.3 ± 2.0 kPa, Es = 2.1 ± 0.75, porcine; HA = 3.1 ± 2.0 kPa, Es = 2.8 ± 1.7, human). Poisson's ratio was estimated to be 0.39 and 0.40 for porcine and human tissue, respectively, from moduli values in a Monte Carlo simulation. To calculate hydraulic permeability, a biphasic finite element model's predictions were numerically matched to experimental data for the time-varying ramp and hold phase of a single increment of applied strain (k = 7.4 ± 4.1 × 10-15 m4/N.s, porcine; k = 7.4 ± 4.3 × 10-15 m4/N.s, human). We can use these newly measured properties to predict fluid flow gradients across the tissue in response to previously reported intra-articular pressures. These values for material constants are to our knowledge the first available measurements in synovium that are necessary to better understand drug transport in both healthy and pathological joints.

Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

Cells of the adult nucleus pulposus (NP) are critically important in maintaining overall disc health and function. NP cells reside in a soft, gelatinous matrix that dehydrates and becomes increasingly fibrotic with age. Such changes result in physical cues of matrix stiffness that may be potent regulators of NP cell phenotype and may contribute to a transition toward a senescent and fibroblastic NP cell with a limited capacity for repair. Here, we investigate the mechanosignaling cues generated from changes in matrix stiffness in directing NP cell phenotype and identify mechanisms that can potentially preserve a biosynthetically active, juvenile NP cell phenotype. Using a laminin-functionalized polyethylene glycol hydrogel, we show that when NP cells form rounded, multicell clusters, they are able to maintain cytosolic localization of myocardin-related transcription factor (MRTF)-A, a coactivator of serum-response factor (SRF), known to promote fibroblast-like behaviors in many cells. Upon preservation of a rounded shape, human NP cells similarly showed cytosolic retention of transcriptional coactivator Yes-associated protein (YAP) and its paralogue PDZ-binding motif (TAZ) with associated decline in activation of its transcription factor TEA domain family member-binding domain (TEAD). When changes in cell shape occur, leading to a more spread, fibrotic morphology associated with stronger F-actin alignment, SRF and TEAD are up-regulated. However, targeted deletion of either cofactor was not sufficient to overcome shape-mediated changes observed in transcriptional activation of SRF or TEAD. Findings show that substrate stiffness-induced promotion of F-actin alignment occurs concomitantly with a flattened, spread morphology, decreased NP marker expression, and reduced biosynthetic activity. This work indicates cell shape is a stronger indicator of SRF and TEAD mechanosignaling pathways than coactivators MRTF-A and YAP/TAZ, respectively, and may play a role in the degeneration-associated loss of NP cellularity and phenotype.-Fearing, B. V., Jing, L., Barcellona, M. N., Witte, S. E., Buchowski, J. M., Zebala, L. P., Kelly, M. P., Luhmann, S., Gupta, M. C., Pathak, A., Setton, L. A. Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

Combined Experimental Approach and Finite Element Modeling of Small Molecule Transport Through Joint Synovium to Measure Effective Diffusivity.

Trans-synovial solute transport plays a critical role in the clearance of intra-articularly (IA) delivered drugs. In this study, we present a computational finite element model (FEM) of solute transport through the synovium validated by experiments on synovial explants. Unsteady diffusion of urea, a small uncharged molecule, was measured through devitalized porcine and human synovium using custom-built diffusion chambers. A multiphasic computational model was constructed and optimized with the experimental data to extract effective diffusivity for urea within the synovium. A monotonic decrease in urea concentration was observed in the donor bath over time, with an effective diffusivity found to be an order of magnitude lower in synovium versus that measured in free solution. Parametric studies incorporating an intimal cell layer with varying thickness and varying effective diffusivities were performed, revealing a dependence of drug clearance kinetics on both parameters. The findings of this study indicate that the synovial matrix impedes urea solute transport out of the joint with little retention of the solute in the matrix.

The role of extracellular matrix elasticity and composition in regulating the nucleus pulposus cell phenotype in the intervertebral disc: a narrative review.

Intervertebral disc (IVD) disorders are a major contributor to disability and societal health care costs. Nucleus pulposus (NP) cells of the IVD exhibit changes in both phenotype and morphology with aging-related IVD degeneration that may impact the onset and progression of IVD pathology. Studies have demonstrated that immature NP cell interactions with their extracellular matrix (ECM) may be key regulators of cellular phenotype, metabolism and morphology. The objective of this article is to review our recent experience with studies of NP cell-ECM interactions that reveal how ECM cues can be manipulated to promote an immature NP cell phenotype and morphology. Findings demonstrate the importance of a soft (<700 Pa), laminin-containing ECM in regulating healthy, immature NP cells. Knowledge of NP cell-ECM interactions can be used for development of tissue engineering or cell delivery strategies to treat IVD-related disorders.

IGF-1 Peptide Mimetic-functionalized Hydrogels Enhance MSC Survival and Immunomodulatory Activity.

Human mesenchymal stem cells (MSCs) have demonstrated promise when delivered to damaged tissue or tissue defects for their cytokine secretion and inflammation modulation behaviors that can promote repair. Insulin-like growth factor 1 (IGF-1) has been shown to augment MSCs' viability and survival and promote their secretion of cytokines that signal to endogenous cells, in the treatment of myocardial infarction, wound healing, and age-related diseases. Biomaterial cell carriers can be functionalized with growth factor-mimetic peptides to enhance MSC function while promoting cell retention and minimizing off-target effects seen with direct administration of soluble growth factors. Here, we functionalized alginate hydrogels with three distinct IGF-1 peptide mimetics and the integrin-binding peptide, cyclic RGD. One IGF-1 peptide mimetic (IGM-3) was found to activate Akt signaling and support survival of serum-deprived MSCs. MSCs encapsulated in alginate hydrogels that presented both IGM-3 and cRGD showed a significant reduction in pro-inflammatory cytokine secretion when challenged with interleukin-1ß. Finally, MSCs cultured within the cRGD/IGM-3 hydrogels were able to blunt pro-inflammatory gene expression of human primary cells from degenerated intervertebral discs. These studies indicate the potential to leverage cell adhesive and IGF-1 growth factor peptide mimetics together to control therapeutic secretory behavior of MSCs. Significance Statement: Insulin-like growth factor 1 (IGF-1) plays a multifaceted role in stem cell biology and may promote proliferation, survival, migration, and immunomodulation for MSCs. In this study, we functionalized alginate hydrogels with integrin-binding and IGF-1 peptide mimetics to investigate their impact on MSC function. Embedding MSCs in these hydrogels enhanced their ability to reduce inflammatory cytokine production and promote anti-inflammatory gene expression in cells from degenerative human intervertebral discs exposed to proteins secreted by the MSC. This approach suggests a new way to retain and augment MSC functionality using IGF-1 peptide mimetics, offering an alternative to co-delivery of cells and high dose soluble growth factors for tissue repair and immune- system modulation.

Grand Challenges at the Interface of Engineering and Medicine.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

A multiphasic model for determination of water and solute transport across the arterial wall: effects of elastic fiber defects.

Transport of solute across the arterial wall is a process driven by both convection and diffusion. In disease, the elastic fibers in the arterial wall are disrupted and lead to altered fluid and mass transport kinetics. A computational mixture model was used to numerically match previously published data of fluid and solute permeation experiments in groups of mouse arteries with genetic (knockout of fibulin-5) or chemical (treatment with elastase) disruption of elastic fibers. A biphasic model of fluid permeation indicated the governing property to be the hydraulic permeability, which was estimated to be 1.52×10-9, 1.01×10-8, and 1.07×10-8 mm4/µN.s for control, knockout, and elastase groups, respectively. A multiphasic model incorporating solute transport was used to estimate effective diffusivities that were dependent on molecular weight, consistent with expected transport behaviors in multiphasic biological tissues. The effective diffusivity for the 4 kDA FITC-dextran solute, but not the 70 or 150 kDa FITC-dextran solutes, was dependent on elastic fiber structure, with increasing values from control to knockout to elastase groups, suggesting that elastic fiber disruption affects transport of lower molecular weight solutes. The model used here sets the groundwork for future work investigating transport through the arterial wall.

Single Cell RNA-Sequence Analyses Reveal Uniquely Expressed Genes and Heterogeneous Immune Cell Involvement in the Rat Model of Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we used single-cell RNA-sequencing analysis of degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at the two- or eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at the single-cell resolution. Unsupervised cluster analysis revealed the presence of four known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at two- and eight-weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes, and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration.

Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues.

OBJECTIVE: Prior reports document macrophage and lymphocyte infiltration with proinflammatory cytokine expression in pathologic intervertebral disc (IVD) tissues. Nevertheless, the role of the Th17 lymphocyte lineage in mediating disc disease remains uninvestigated. We undertook this study to evaluate the immunophenotype of pathologic IVD specimens, including interleukin-17 (IL-17) expression, from surgically obtained IVD tissue and from nondegenerated autopsy control tissue. METHODS: Surgical IVD tissues were procured from patients with degenerative disc disease (n = 25) or herniated IVDs (n = 12); nondegenerated autopsy control tissue was also obtained (n = 8) from the anulus fibrosus and nucleus pulposus regions. Immunohistochemistry was performed for cell surface antigens (CD68 for macrophages, CD4 for lymphocytes) and various cytokines, with differences in cellularity and target immunoreactivity scores analyzed between surgical tissue groups and between autopsy control tissue regions. RESULTS: Immunoreactivity for IL-4, IL-6, IL-12, and interferon-gamma (IFNgamma) was modest in surgical IVD tissue, although expression was higher in herniated IVD samples and virtually nonexistent in control samples. The Th17 lymphocyte product IL-17 was present in >70% of surgical tissue fields, and among control samples was detected rarely in anulus fibrosus regions and modestly in nucleus pulposus regions. Macrophages were prevalent in surgical tissues, particularly herniated IVD samples, and lymphocytes were expectedly scarce. Control tissue revealed lesser infiltration by macrophages and a near absence of lymphocytes. CONCLUSION: Greater IFNgamma positivity, macrophage presence, and cellularity in herniated IVDs suggests a pattern of Th1 lymphocyte activation in this pathology. Remarkable pathologic IVD tissue expression of IL-17 is a novel finding that contrasts markedly with low levels of IL-17 in autopsy control tissue. These findings suggest involvement of Th17 lymphocytes in the pathomechanism of disc degeneration.

Size-Dependent Effective Diffusivity in Healthy Human and Porcine Joint Synovium.

Intra-articular drug delivery can be effective in targeting a diseased joint but is hampered by rapid clearance times from the diarthrodial joint. The synovium is a multi-layered tissue that surrounds the diarthrodial joint and governs molecular transport into and out of the joint. No models of drug clearance through synovium exist to quantify diffusivity across solutes, tissue type and disease pathology. We previously have developed a finite element model of synovium as a porous, permeable, fluid-filled tissue and used an inverse method to determine urea's effective diffusivity (Deff) in de-vitalized synovium explants.22 Here we apply this method to determine Deff from unsteady diffusive transport of model solutes and confirm the role of molecular weight in solute transport. As molecular weight increased, Deff decreased in both human and porcine tissues, with similar behavior across the two species. Unsteady transport was well-described by a single exponential transient decay in concentration, yielding solute half-lives (t1/2) that compared favorably with the Deff determined from the finite element model fit. Determined values for Deff parallel prior observations of size-dependent in vivo drug clearance and provide an intrinsic parameter with greater ability to resolve size-dependence in vitro. Thus, this work forms the basis for understanding the influence of size on drug transport in synovium and can guide future studies to elucidate the role of charge and tissue pathology on the transport of therapeutics in healthy and pathological human synovium.

Bioactive in situ crosslinkable polymer-peptide hydrogel for cell delivery to the intervertebral disc in a rat model.

Degeneration of the intervertebral disc (IVD) is associated with significant biochemical and morphological changes that include a loss of disc height, decreased water content and decreased cellularity. Cell delivery has been widely explored as a strategy to supplement the nucleus pulposus (NP) region of the degenerated IVD in both pre-clinical and clinical trials, using progenitor or primary cell sources. We previously demonstrated an ability for a polymer-peptide hydrogel, serving as a culture substrate, to promote adult NP cells to undergo a shift from a degenerative fibroblast-like state to a juvenile-like NP phenotype. In the current study, we evaluate the ability for this peptide-functionalized hydrogel to serve as a bioactive system for cell delivery, retention and preservation of a biosynthetic phenotype for primary IVD cells delivered to the rat caudal disc in an anular puncture degeneration model. Our data suggest that encapsulation of adult degenerative human NP cells in a stiff formulation of the hydrogel functionalized with laminin-mimetic peptides IKVAV and AG73 can promote cell viability and increased biosynthetic activity for this population in 3D culture in vitro. Delivery of the peptide-functionalized biomaterial with primary rat cells to the degenerated IVD supported NP cell retention and NP-specific protein expression in vivo, and promoted improved disc height index (DHI) values and endplate organization compared to untreated degenerated controls. The results of this study suggest the physical cues of this peptide-functionalized hydrogel can serve as a supportive carrier for cell delivery to the IVD. STATEMENT OF SIGNIFICANCE: Cell delivery into the degenerative intervertebral disc has been widely explored as a strategy to supplement the nucleus pulposus. The current work seeks to employ a biomaterial functionalized with laminin-mimetic peptides as a cell delivery scaffold in order to improve cell retention rates within the intradiscal space, while providing the delivered cells with biomimetic cues in order to promote phenotypic expression and increase biosynthetic activity. The use of the in situ crosslinkable material integrated with the native IVD, presenting a system with adequate physical properties to support a degenerative disc.

Immunoengineering the next generation of arthritis therapies.

Immunoengineering continues to revolutionize healthcare, generating new approaches for treating previously intractable diseases, particularly in regard to cancer immunotherapy. In joint diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA), biomaterials and anti-cytokine treatments have previously been at that forefront of therapeutic innovation. However, while many of the existing anti-cytokine treatments are successful for a subset of patients, these treatments can also pose severe risks, adverse events and off-target effects due to continuous delivery at high dosages or a lack of disease-specific targets. The inadequacy of these current treatments has motivated the development of new immunoengineering strategies that offer safer and more efficacious alternative therapies through the precise and controlled targeting of specific upstream immune responses, including direct and mechanistically-driven immunoengineering approaches. Advances in the understanding of the immunomodulatory pathways involved in musculoskeletal disease, in combination with the growing emphasis on personalized medicine, stress the need for carefully considering the delivery strategies and therapeutic targets when designing therapeutics to better treat RA and OA. Here, we focus on recent advances in biomaterial and cell-based immunomodulation, in combination with genetic engineering, for therapeutic applications in joint diseases. The application of immunoengineering principles to the study of joint disease will not only help to elucidate the mechanisms of disease pathogenesis but will also generate novel disease-specific therapeutics by harnessing cellular and biomaterial responses. STATEMENT OF SIGNIFICANCE: It is now apparent that joint diseases such as osteoarthritis and rheumatoid arthritis involve the immune system at both local (i.e., within the joint) and systemic levels. In this regard, targeting the immune system using both biomaterial-based or cellular approaches may generate new joint-specific treatment strategies that are well-controlled, safe, and efficacious. In this review, we focus on recent advances in immunoengineering that leverage biomaterials and/or genetically engineered cells for therapeutic applications in joint diseases. The application of such approaches, especially synergistic strategies that target multiple immunoregulatory pathways, has the potential to revolutionize our understanding, treatment, and prevention of joint diseases.

Electric Field Stimulation for the Functional Assessment of Isolated Dorsal Root Ganglion Neuron Excitability.

Genetically encoded calcium indicators have proven useful for characterizing dorsal root ganglion neuron excitability in vivo. Challenges persist in achieving high spatial-temporal resolutions in vivo, however, due to deep tissue imaging and motion artifacts that may be limiting technical factors in obtaining measurements. Here we report an ex vivo imaging method, using a peripheral neuron-specific Advillin-GCaMP mouse line and electric field stimulation of dorsal root ganglion tissues, to assess the sensitivity of neurons en bloc. The described method rapidly characterizes Ca2+ activity in hundreds of dorsal root ganglion neurons (221 ± 64 per dorsal root ganglion) with minimal perturbation to the in situ soma environment. We further validate the method for use as a drug screening platform with the voltage-gated sodium channel inhibitor, tetrodotoxin. Drug treatment led to decreased evoked Ca2+ activity; half-maximal response voltage (EV50) increased from 13.4 V in untreated tissues to 21.2, 23.3, 51.5 (p < 0.05), and 60.6 V (p < 0.05) at 0.01, 0.1, 1, and 10 µM doses, respectively. This technique may help improve an understanding of neural signaling while retaining tissue structural organization and serves as a tool for the rapid ex vivo recording and assessment of neural activity.

Development of a library of laminin-mimetic peptide hydrogels for control of nucleus pulposus cell behaviors.

The nucleus pulposus (NP) of the intervertebral disc plays a critical role in distributing mechanical loads to the axial skeleton. Alterations in NP cells and, consequently, NP matrix are some of the earliest changes in the development of disc degeneration. Previous studies demonstrated a role for laminin-presenting biomaterials in promoting a healthy phenotype for human NP cells from degenerated tissue. Here we investigate the use of laminin-mimetic peptides presented individually or in combination on a poly(ethylene) glycol hydrogel as a platform to modulate the behaviors of degenerative human NP cells. Data confirm that NP cells attach to select laminin-mimetic peptides that results in cell signaling downstream of integrin and syndecan binding. Furthermore, the peptide-functionalized hydrogels demonstrate an ability to promote cell behaviors that mimic that of full-length laminins. These results identify a set of peptides that can be used to regulate NP cell behaviors toward a regenerative engineering strategy.

Integrin and syndecan binding peptide-conjugated alginate hydrogel for modulation of nucleus pulposus cell phenotype.

Biomaterial based strategies have been widely explored to preserve and restore the juvenile phenotype of cells of the nucleus pulposus (NP) in degenerated intervertebral discs (IVD). With aging and maturation, NP cells lose their ability to produce necessary extracellular matrix and proteoglycans, accelerating disc degeneration. Previous studies have shown that integrin or syndecan binding peptide motifs from laminin can induce NP cells from degenerative human discs to re-express juvenile NP-specific cell phenotype and biosynthetic activity. Here, we engineered alginate hydrogels to present integrin- and syndecan-binding peptides alone or in combination (cyclic RGD and AG73, respectively) to introduce bioactive features into the alginate gels. We demonstrated human NP cells cultured upon and within alginate hydrogels presented with cRGD and AG73 peptides exhibited higher cell viability, biosynthetic activity, and NP-specific protein expression over alginate alone. Moreover, the combination of the two peptide motifs elicited markers of the NP-specific cell phenotype, including N-Cadherin, despite differences in cell morphology and multicellular cluster formation between 2D and 3D cultures. These results represent a promising step toward understanding how distinct adhesive peptides can be combined to guide NP cell fate. In the future, these insights may be useful to rationally design hydrogels for NP cell-transplantation based therapies for IVD degeneration.

Control of adhesive ligand density for modulation of nucleus pulposus cell phenotype.

Cells of the nucleus pulposus have been observed to undergo a shift from their notochordal-like juvenile phenotype to a more fibroblast-like state with age and maturation. It has been demonstrated that culture of degenerative adult human nucleus pulposus cells upon soft (<1 kPa) full length laminin-containing hydrogel substrates promotes increased levels of a panel of markers associated with the juvenile nucleus pulposus cell phenotype. In the current work, we observed an ability to use soft polymeric substrates functionalized with short laminin-mimetic peptide sequences to recapitulate the behaviors elicited by soft, full-length laminin containing materials. Furthermore, our work suggests an ability to mimic features of soft systems through control of peptide density upon stiffer substrates. Specifically, results suggest that stiffer polymer-peptide hydrogel substrates can be used to promote the expression of a more juvenile-like phenotype for cells of the nucleus pulposus by reducing adhesive ligand presentation. Here we show how polymer stiffness combined with adhesive ligand presentation can be controlled to be supportive of nucleus pulposus cell phenotype and biosynthesis.

Verteporfin treatment controls morphology, phenotype, and global gene expression for cells of the human nucleus pulposus.

Cells of the nucleus pulposus (NP) are essential contributors to extracellular matrix synthesis and function of the intervertebral disc. With age and degeneration, the NP becomes stiffer and more dehydrated, which is associated with a loss of phenotype and biosynthetic function for its resident NP cells. Also, with aging, the NP cell undergoes substantial morphological changes from a rounded shape with pronounced vacuoles in the neonate and juvenile, to one that is more flattened and spread with a loss of vacuoles. Here, we make use of the clinically relevant pharmacological treatment verteporfin (VP), previously identified as a disruptor of yes-associated protein-TEA domain family member-binding domain (TEAD) signaling, to promote morphological changes in adult human NP cells in order to study variations in gene expression related to differences in cell shape. Treatment of adult, degenerative human NP cells with VP caused a shift in morphology from a spread, fibroblastic-like shape to a rounded, clustered morphology with decreased transcriptional activity of TEAD and serum-response factor. These changes were accompanied by an increased expression of vacuoles, NP-specific gene markers, and biosynthetic activity. The contemporaneous observation of VP-induced changes in cell shape and prominent, time-dependent changes within the transcriptome of NP cells occurred over all timepoints in culture. Enriched gene sets with the transition to VP-induced cell rounding suggest a major role for cell adhesion, cytoskeletal remodeling, vacuolar lumen, and MAPK activity in the NP phenotypic and functional response to changes in cell shape.