Extracellular Vesicles Generated by Mesenchymal Stem Cells in Stirred Suspension Bioreactors Promote Angiogenesis in Human-Brain-Derived Endothelial Cells.

Interrupted blood flow in the brain due to ischemic injuries such as ischemic stroke or traumatic brain injury results in irreversible brain damage, leading to cognitive impairment associated with inflammation, disruption of the blood-brain barrier (BBB), and cell death. Since the BBB only allows entry to a small class of drugs, many drugs used to treat ischemia in other tissues have failed in brain-related disorders. The administration of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) has shown promise in improving the functional recovery of the brain following cerebral ischemia by inducing blood vessel formation. To facilitate such a treatment approach, it is necessary to develop bioprocesses that can produce therapeutically relevant MSC-EVs in a reproducible and scalable manner. This study evaluated the feasibility of using stirred suspension bioreactors (SSBs) to scale-up the serum-free production of pro-angiogenic MSC-EVs under clinically relevant physioxic conditions. It was found that MSCs grown in SSBs generated EVs that stimulated angiogenesis in cerebral microvascular endothelial cells, supporting the use of SSBs to produce MSC-EVs for application in cerebral ischemia. These properties were impaired at higher cell confluency, outlining the importance of considering the time of harvest when developing bioprocesses to manufacture EV populations.

Physiological oxygen conditions enhance the angiogenic properties of extracellular vesicles from human mesenchymal stem cells.

BACKGROUND: Following an ischemic injury to the brain, the induction of angiogenesis is critical to neurological recovery. The angiogenic benefits of mesenchymal stem cells (MSCs) have been attributed at least in part to the actions of extracellular vesicles (EVs) that they secrete. EVs are membrane-bound vesicles that contain various angiogenic biomolecules capable of eliciting therapeutic responses and are of relevance in cerebral applications due to their ability to cross the blood-brain barrier (BBB). Though MSCs are commonly cultured under oxygen levels present in injected air, when MSCs are cultured under physiologically relevant oxygen conditions (2-9% O2), they have been found to secrete higher amounts of survival and angiogenic factors. There is a need to determine the effects of MSC-EVs in models of cerebral angiogenesis and whether those from MSCs cultured under physiological oxygen provide greater functional effects. METHODS: Human adipose-derived MSCs were grown in clinically relevant serum-free medium and exposed to either headspace oxygen concentrations of 18.4% O2 (normoxic) or 3% O2 (physioxic). EVs were isolated from MSC cultures by differential ultracentrifugation and characterized by their size, concentration of EV specific markers, and their angiogenic protein content. Their functional angiogenic effects were evaluated in vitro by their induction of cerebral microvascular endothelial cell (CMEC) proliferation, tube formation, and angiogenic and tight junction gene expressions. RESULTS: Compared to normoxic conditions, culturing MSCs under physioxic conditions increased their expression of angiogenic genes SDF1 and VEGF, and subsequently elevated VEGF-A content in the EV fraction. MSC-EVs demonstrated an ability to induce CMEC angiogenesis by promoting tube formation, with the EV fraction from physioxic cultures having the greatest effect. The physioxic EV fraction further upregulated the expression of CMEC angiogenic genes FGF2, HIF1, VEGF and TGFB1, as well as genes (OCLN and TJP1) involved in BBB maintenance. CONCLUSIONS: EVs from physioxic MSC cultures hold promise in the generation of a cell-free therapy to induce angiogenesis. Their positive angiogenic effect on cerebral microvascular endothelial cells demonstrates that they may have utility in treating ischemic cerebral conditions, where the induction of angiogenesis is critical to improving recovery and neurological function.

Exogenously delivered iPSCs disrupt the natural repair response of endogenous MPCs after bone injury.

Promoting bone healing including fracture non-unions are promising targets for bone tissue engineering due to the limited success of current clinical treatment methods. There has been significant research on the use of stem cells with and without biomaterial scaffolds to treat bone fractures due to their promising regenerative capabilities. However, the relative roles of exogenous vs. endogenous stem cells and their overall contribution to in vivo fracture repair is not well understood. The purpose of this study was to determine the interaction between exogenous and endogenous stem cells during bone healing. This study was conducted using a standardized burr-hole bone injury model in a mesenchymal progenitor cell (MPC) lineage-tracing mouse under normal homeostatic and osteoporotic conditions. Burr-hole injuries were treated with a collagen-I biomaterial loaded with and without labelled induced pluripotent stem cells (iPSCs). Using lineage-tracing, the roles of exogenous and endogenous stem cells during bone healing were examined. It was observed that treatment with iPSCs resulted in muted healing compared to untreated controls in intact mice post-injury. When the cell populations were examined histologically, iPSC-treated burr-hole defects presented with a dramatic reduction in endogenous MPCs and cell proliferation throughout the injury site. However, when the ovaries were removed and an osteoporotic-like phenotype induced in the mice, iPSCs treatment resulted in increased bone formation relative to untreated controls. In the absence of iPSCs, endogenous MPCs demonstrated robust proliferative and osteogenic capacity to undertake repair and this behaviour was disrupted in the presence of iPSCs which instead took on an osteoblast fate but with little proliferation. This study clearly demonstrates that exogenously delivered cell populations can impact the normal function of endogenous stem/progenitor populations during the normal healing cascade. These interactions need to be better understood to inform cell and biomaterial therapies to treat fractures.

Production of Mesenchymal Progenitor Cell-Derived Extracellular Vesicles in Suspension Bioreactors for Use in Articular Cartilage Repair.

Mesenchymal progenitor cells (MPCs) have shown promise initiating articular cartilage repair, with benefits largely attributed to the trophic factors they secrete. These factors can be found in the conditioned medium (CM) collected from cell cultures, and it is believed that extracellular vesicles (EVs) within this CM are at least partially responsible for MPC therapeutic efficacy. This study aimed to examine the functionality of the EV fraction of CM compared to whole CM obtained from human adipose-derived MPCs in an in vivo murine cartilage defect model. Mice treated with whole CM or the EV fraction demonstrated an enhanced cartilage repair score and type II collagen deposition at the injury site compared to saline controls. We then developed a scalable bioprocess using stirred suspension bioreactors (SSBs) to generate clinically relevant quantities of MPC-EVs. Whereas static monolayer culture systems are simple to use and readily accessible, SSBs offer increased scalability and a more homogenous environment due to constant mixing. This study evaluated the biochemical and functional properties of MPCs and their EV fractions generated in static culture versus SSBs. Functionality was assessed using in vitro MPC chondrogenesis as an outcome measure. SSBs supported increased MPC expression of cartilage-specific genes, and EV fractions derived from both static and SSB culture systems upregulated type II collagen production by MPCs. These results suggest that SSBs are an effective platform for the generation of MPC-derived EVs with the potential to induce cartilage repair.

Pre-Columbian fire management and control of climate-driven floodwaters over 3,500 years in southwestern Amazonia.

In landscapes that support economic and cultural activities, human communities actively manage environments and environmental change at a variety of spatial scales that complicate the effects of continental-scale climate. Here, we demonstrate how hydrological conditions were modified by humans against the backdrop of Holocene climate change in southwestern Amazonia. Paleoecological investigations (phytoliths, charcoal, pollen, diatoms) of two sediment cores extracted from within the same permanent wetland, ∼22 km apart, show a 1,500-y difference in when the intensification of land use and management occurred, including raised field agriculture, fire regime, and agroforestry. Although rising precipitation is well known during the mid to late Holocene, human actions manipulated climate-driven hydrological changes on the landscape, revealing differing histories of human landscape domestication. Environmental factors are unable to account for local differences without the mediation of human communities that transformed the region to its current savanna/forest/wetland mosaic beginning at least 3,500 y ago. Regional environmental variables did not drive the choices made by farmers and fishers, who shaped these local contexts to better manage resource extraction. The savannas we observe today were created in the post-European period, where their fire regime and structural diversity were shaped by cattle ranching.

Repetitive in vivo manual loading of the spine elicits cellular responses in porcine annuli fibrosi.

Back pain and intervertebral disc degeneration are prevalent, costly, and widely treated by manual therapies, yet the underlying causes of these diseases are indeterminate as are the scientific bases for such treatments. The present studies characterize the effects of repetitive in vivo manual loads on porcine intervertebral disc cell metabolism using RNA deep sequencing. A single session of repetitive manual loading applied to the lumbar spine induced both up- and down-regulation of a variety of genes transcribed by cells in the ventral annuli fibrosi. The effect of manual therapy at the level of loading was greater than at a level distant to the applied load. Gene ontology and molecular pathway analyses categorized biological, molecular, and cellular functions influenced by repetitive manual loading, with over-representation of membrane, transmembrane, and pericellular activities. Weighted Gene Co-expression Network Analysis discerned enrichment in genes in pathways of inflammation and skeletogenesis. The present studies support previous findings of intervertebral disc cell mechanotransduction, and are the first to report comprehensively on the repertoire of gene targets influenced by mechanical loads associated with manual therapy interventions. The present study defines the cellular response of repeated, low-amplitude loads on normal healthy annuli fibrosi and lays the foundation for future work defining how healthy and diseased intervertebral discs respond to single or low-frequency manual loads typical of those applied clinically.

Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics.

Mesenchymal stem cells (MSCs) have attracted tremendous research interest due to their ability to repair tissues and reduce inflammation when implanted into a damaged or diseased site. These therapeutic effects have been largely attributed to the collection of biomolecules they secrete (i.e., their secretome). Recent studies have provided evidence that similar effects may be produced by utilizing only the secretome fraction containing extracellular vesicles (EVs). EVs are cell-derived, membrane-bound vesicles that contain various biomolecules. Due to their small size and relative mobility, they provide a stable mechanism to deliver biomolecules (i.e., biological signals) throughout an organism. The use of the MSC secretome, or its components, has advantages over the implantation of the MSCs themselves: (i) signals can be bioengineered and scaled to specific dosages, and (ii) the nonliving nature of the secretome enables it to be efficiently stored and transported. However, since the composition and therapeutic benefit of the secretome can be influenced by cell source, culture conditions, isolation methods, and storage conditions, there is a need for standardization of bioprocessing parameters. This review focuses on key parameters within the MSC culture environment that affect the nature and functionality of the secretome. This information is pertinent to the development of bioprocesses aimed at scaling up the production of secretome-derived products for their use as therapeutics.

Effect of mechanical strain on the pluripotency of murine embryonic stem cells seeded in a collagen-I scaffold.

The use of embryonic stem cells (ESC) in regenerative medicine is restricted due to the possibility of tumorigenicity after inefficient or incomplete differentiation. Studies from our group, and others, suggest that mechanical stimuli may have a suppressive effect on the pluripotency/tumorigenesis of murine ESC (mESC). Furthermore, we have demonstrated that mESC seeded in a type I collagen scaffold, and transplanted into a murine bone fracture model, demonstrated repair without tumor formation. However, it remains unknown if mechanical factors were involved in blocking tumorigenicity of the mESC. Therefore, the aims of the current study were: (i) to characterize the mechanical environment within the transplanted construct (mESC-Col I) in an in vivo murine fracture model using computational analyses; and (ii) to reproduce this mechanical environment in vitro to elucidate the role of these mechanical factors on mESC pluripotent gene expression. It was predicted that the mESC-Col I construct was subjected to an average octahedral shear strain of ∼3.8% and a compressive strain of ∼3.1% within the fracture in vivo when the murine tibia was subjected to an axial compression load of 4 N (1 Hz). When a similar strain environment was replicated experimentally in vitro, the expression patterns of marker genes for pluripotency (Oct 4, Sox 2, Nanog, Rex 1, and oncogene ERas) were significantly down-regulated. This suggests that the local micro-mechanical environment within the fracture site in vivo may be involved in regulating stem cell fate after transplantation, and that these physical factors should be considered when developing regenerative medicine strategies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:799-807, 2018.

Reduction of pluripotent gene expression in murine embryonic stem cells exposed to mechanical loading or Cyclo RGD peptide.

BACKGROUND: Self-renewal and differentiation of embryonic stem cells (ESCs) is directed by biological and/or physical cues that regulate multiple signaling cascades. We have previously shown that mESCs seeded in a type I collagen matrix demonstrate a loss of pluripotent marker expression and differentiate towards an osteogenic lineage. In this study, we examined if this effect was mediated in part through Arginylglycylaspartic acid (RGD) dependent integrin activity and/or mechano-transduction. RESULTS: The results from this study suggest that mESC interaction with the local microenvironment through RGD dependent integrins play a role in the regulation of mESC core transcription factors (TF), Oct-4, Sox 2 and Nanog. Disruption of this interaction with a cyclic RGD peptide (cRGDfC) was sufficient to mimic the effect of a mechanical stimulus in terms of pluripotent gene expression, specifically, we observed that supplementation with cRGDfC, or mechanical stimulus, significantly influenced mESC pluripotency by down-regulating core transcription factors. Moreover, our results indicated that the presence of the cRGDfC peptide inhibited integrin expression and up-regulated early lineage markers (mesoderm and ectoderm) in a Leukemia inhibitory factor (LIF) dependent manner. When cRGDfC treated mESCs were injected in Severe combined immunodeficiency (SCID) mice, no tissue growth and/or teratoma formation was observed, suggesting that the process of mESC tumor formation in vivo is potentially dependent on integrin interaction. CONCLUSIONS: Overall, the disruption of cell-integrin interaction via cRGDfC peptide can mimic the effect of mechanical stimulation on mESC pluripotency gene expression and also inhibit the tumorigenic potential of mESCs in vivo.

The role of gap junctions and mechanical loading on mineral formation in a collagen-I scaffold seeded with osteoprogenitor cells.

Fracture nonunions represent one of many large bone defects where current treatment strategies fall short in restoring both form and function of the injured tissue. In this case, the use of a tissue-engineered scaffold for promoting bone healing offers an accessible and easy-to-manipulate environment for studying bone formation processes in vitro. We have previously shown that mechanical prestimulation using confined compression of differentiating osteoblasts results in an increase in mineralization formed in a 3D collagen-I scaffold. This study builds on this knowledge by evaluating the short and long-term effects of blocking gap junction-mediated intercellular communication among osteogenic cells on their effectiveness to mineralize collagen-I scaffolds in vitro, and in the presence and absence of mechanical stimulation. In this study, confined compression was applied in conjunction with octanol (a general communication blocker) or 18-α-glycerrhetinic acid (AGA, a specific gap junction blocker) using a modified FlexCell plate to collagen-I scaffolds seeded with murine embryonic stem cells stimulated toward osteoblast differentiation using beta-glycerol phosphate. The activity, presence, and expression of osteoblast cadherin, connexin-43, as well as various pluripotent and osteogenic markers were examined at 5-30 days of differentiation. Fluorescence recovery after photobleaching, immunofluorescence, viability, histology assessments, and reverse-transcriptase polymerase chain reaction assessments revealed that inhibiting communication in this scaffold altered the lineage and function of differentiating osteoblasts. In particular, treatment with communication inhibitors caused reduced mineralization in the matrix, and dissociation between connexin-43 and integrin α5ß1. This dissociation was not restored even after long-term recovery. Thus, in order for this scaffold to be considered as an alternative strategy for the repair of large bone defects, cell-cell contacts and cell-matrix interactions must remain intact for osteoblast differentiation and function to be preserved. This study shows that within this 3D scaffold, gap junctions are essential in osteoblast response to mechanical loading, and are essential structures in producing a significant amount and organization of mineralization in the matrix.

The effect of mechanical stimulation on mineralization in differentiating osteoblasts in collagen-I scaffolds.

Developing a viable and functional bone scaffold in vitro that is capable of surviving and bearing mechanical load in vivo requires an understanding of the cell biology of osteoprogenitor cells, particularly how they are influenced by mechanical stimulation during cell differentiation and maturation. In this study, mechanical load was applied using a modified FlexCell plate to impart confined compression to collagen-I scaffolds seeded with undifferentiated murine embryonic stem cells. The activity, presence, and expression of osteoblast-cadherin (OB-Cad) and connexin-43, as well as various pluripotent and osteogenic markers were examined at 5-30 days of differentiation as cells were stimulated to differentiate to osteoblasts with and without applied mechanical load. Fluorescence recovery after photobleaching, immunofluorescence, viability, von Kossa, and real-time polymerase chain reaction assessments revealed that mechanical prestimulation of this cell-seeded scaffold altered the expression of OB-Cad and connexin-43 and resulted in significant differences in the structure and organization of mineralization present in the collagen matrix. Specifically, cells in gels that were loaded for 40 h after 5 days of differentiation and then left to fully differentiate for 30 days produced a highly structured honeycomb-shaped mineralization in the matrix; an outcome that was previously shown to be indicative of late osteoblast/early osteocyte activity. This study highlights the potential of mechanical load to accelerate differentiation and enhance osteoblast communication and function during the differentiation process, and highlights a time point of cell differentiation within this scaffold to apply load in order to most effectively transduce a mechanical signal.

In situ cell-matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials.

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell-matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell-matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (∼2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell-matrix boundary with visible shear across cell nuclei of >1 µm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus.

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell-cell and cell-matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.

Serum-free media for articular chondrocytes in vitro expansion.

BACKGROUND: In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. METHODS: Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining. RESULTS: Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium. CONCLUSION: These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

Strain transfer in the annulus fibrosus under applied flexion.

A detailed understanding of the anatomical and mechanical environment in the intervertebral disc at the scale of the cell is necessary for the design of tissue engineering repair strategies and to elucidate the role of mechanical factors in pathology. The objective of this study was to measure and compare the macroscale to microscale strains in the outer annulus fibrosus in various cellular regions of intact discs over a range of applied flexion. Macroscale strains were measured on the annulus fibrosus surface, and contrasted to in situ microscale strains using novel confocal microscopy techniques for dual labeling of the cell and the extracellular matrix. Fiber oriented surface strains were significantly higher than in situ fiber strains, which implies a mechanism of load redistribution that minimizes strain along the fibers. Non-uniformity of the strains and matrix distortion occurred immediately and most interestingly varied little with increase in flexion (3-16 degrees), suggesting that inter-fiber shear is important in the initial stages of strain redistribution. Fiber oriented intercellular strains were significantly larger and compressive compared to in situ strains in other regions of the extracellular matrix indicating that the mechanical environment in this region may be unique. Further examination of the structural morphology in this pericellular region is needed to fully understand the pathway of strain transfer from the tissue to the cell. This study provides new knowledge on the complex in situ micro-mechanical environment of the annulus fibrosus that is essential to understanding the mechanobiological behavior of this tissue.

Gourd and squash artifacts yield starch grains of feasting foods from preceramic Peru.

In a study of residues from gourd and squash artifacts, we recovered starch grains from manioc (Manihot esculenta), potato (Solanum sp.), chili pepper (Capsicum spp.), arrowroot (Maranta arundinacea), and algarrobo (Prosopis sp.) from feasting contexts at the Buena Vista site, a central Peruvian preceramic site dating to approximately 2200 calendar years B.C. This study has implications for the study of plant food use wherever gourds or squashes are preserved, documents the earliest evidence for the consumption of algarrobo and arrowroot in Peru, and provides insights into foods consumed at feasts.

Cell deformation and micromechanical environment in the intervertebral disc.

The purpose of this research was to explore the in situ anatomic and mechanical environment of disc cells. Laser scanning confocal microscopy was used to characterize three-dimensional morphology of intervertebral disc cells, micromechanical deformation and interaction with extracellular matrix, and functional intercellular communication. Bovine coccygeal discs were used for both the anatomic and micromechanical investigations. Anulus fibrosus cells had a complex morphology with sinuous processes woven into the extracellular matrix, particularly in the outer aspect of the anulus where they were also interconnected via functional gap junctions. They were also found in an extensive pericellular matrix of type-VI collagen, joining as many as ten cells into linear cell arrays that could be extracted from the matrix as functional units. Mechanically, collagen fibril sliding was demonstrated to govern cell mechanics and strain transfer in the anulus fibrosus during loading activities. Lamellar cells were largely protected from direct tensile strains in the matrix, with minimal intercellular strains. However, intercellular strains between lamellar cells in adjacent arrays were large, illustrating shearing between linear cell arrays. Appreciable shear was observed across the lamellar cell bodies as well as to the cellular processes woven into the matrix. These findings demonstrated the morphologic and micromechanical complexity of anulus fibrosus cells. The knowledge of the in situ environment of disc cells will provide a base to investigate the mechanical implications of disc degeneration on the cellular environment and to better understand how mechanical and genetic risk factors can impact the cells that are essential to maintaining the intervertebral disc.

Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison.

The nuclei pulposi of the intervertebral discs (IVDs) contain a mixed population of cell types at various stages of maturation. This tissue is formed either by or with the help of cells from the embryonic notochord, which appear to be replaced during development by a population of chondrocyte-like cells of uncertain origin. However, this transition occurs at widely varying times, depending upon the species--or even breed--of the animal being examined. There is considerable debate among spine researchers as to whether the presence of these residual notochordal cells has a significant impact upon IVD degeneration models, and thus which models may best represent the human condition. The present study examines several different species commonly used in lumbar spine investigations to explore the variability of notochordal cells in the IVD.

ISSLS prize winner: Collagen fibril sliding governs cell mechanics in the anulus fibrosus: an in situ confocal microscopy study of bovine discs.

STUDY DESIGN: In situ investigation of collagen and cell mechanics in bovine caudal discs using novel techniques of confocal microscopy. OBJECTIVE: To measure simultaneously the in situ intercellular and collagen matrix mechanics in the inner and outer anulus fibrosus of the intervertebral disc subjected to flexion. SUMMARY OF BACKGROUND DATA: Mechanobiology studies, both in vivo and in vitro, clearly demonstrate that mechanical factors can influence the metabolic activity of disc cells, altering the expression of key extracellular matrix molecules. Essential to elucidating the mechanotransduction mechanisms is a detailed understanding of the in situ mechanical environment of disc cells in response to whole-body mechanical loads. METHODS: Confocal microscopy was used to simultaneously track and capture in situ images of fluorescently labeled cells and matrix during an applied flexion. The position of the nuclear centroids was calculated before and after applied flexion to quantify the in situ intercellular mechanics of both lamellar and interlamellar cells. The deflection patterns of lines photobleached into the extracellular matrix were used to quantify collagen fibril sliding and collagen fibril strains in situ. RESULTS: The extracellular matrix was observed to deflect nonuniformly due to the relative sliding of the collagen fibrils. Intercellular displacements within the lamellar layers were also nonuniform, both along a cell row and between adjacent rows. Within a cell row, the intercellular displacements were small (<1%). CONCLUSIONS: The in situ cell mechanics of anular cells was found to be strongly influenced by collagen fibril sliding in the extracellular matrix and could not be inferred directly from applied tissue loads.

The functional significance of cell clusters in the notochordal nucleus pulposus: survival and signaling in the canine intervertebral disc.

STUDY DESIGN: Cell viability was assessed in relation to cell clustering, and mechanisms of cell-cell signaling in the clusters were investigated. OBJECTIVES: To explore the functional role of cell clustering in the notochordal nucleus pulposus. SUMMARY OF BACKGROUND DATA: The intervertebral disc of some species contains residual cells from the embryonic notochord. These cells form large three-dimensional clusters in the young, healthy disc but are replaced by chondrocyte-like cells during aging and degeneration. METHODS: Forty nucleus pulposi of adult dog lumbar intervertebral discs were isolated, and were left untreated, mechanically disrupted through a syringe, or enzymatically digested. The presence of functional gap junctions was determined by the fluorescence recovery after photobleaching method. Cell viability was also assessed over 20 days in vitro. RESULTS: The cell clusters were interconnected via functional gap junctions. Mechanical disruption of the tissue had little effect on long-term cell viability, but enzymatic disruption of the tissue had a substantial negative impact on cell survival. CONCLUSIONS: These results demonstrate that the notochordal cells in adult dog nucleus pulposi are able to communicate via cytoplasmic signals and that such communications may influence the functionality of these cells in the young disc.