Decreased SIRT1 Activity Is Involved in the Acute Injury Response of Chondrocytes to Ex Vivo Injurious Mechanical Overload.

A better understanding of molecular events following cartilage injury is required to develop treatments that prevent or delay the onset of trauma-induced osteoarthritis. In this study, alterations to SIRT1 activity in bovine articular cartilage explants were evaluated in the 24 h following a mechanical overload, and the effect of pharmacological SIRT1 activator SRT1720 on acute chondrocyte injury was assessed. SIRT1 enzymatic activity decreased as early as 5 min following the mechanical overload, and remained suppressed for at least 24 h. The chondrocyte injury response, including apoptosis, oxidative stress, secretion of inflammatory mediators, and alterations in cartilage matrix expression, was prevented with pharmacological activation of SIRT1 in a dose-dependent manner. Overall, the results implicate SIRT1 deactivation as a key molecular event in chondrocyte injury following a mechanical impact overload. As decreased SIRT1 signaling is associated with advanced age, these findings suggest that downregulated SIRT1 activity may be common to both age-related and injury-induced osteoarthritis.

Leaf herbivory by insects during summer reduces overwinter browsing by moose.

BACKGROUND: Damage to plants by herbivores potentially affects the quality and quantity of the plant tissue available to other herbivore taxa that utilize the same host plants at a later time. This study addresses the indirect effects of insect herbivores on mammalian browsers, a particularly poorly-understood class of interactions. Working in the Alaskan boreal forest, we investigated the indirect effects of insect damage to Salix interior leaves during the growing season on the consumption of browse by moose during winter, and on quantity and quality of browse production. RESULTS: Treatment with insecticide reduced leaf mining damage by the willow leaf blotch miner, Micrurapteryx salicifoliella, and increased both the biomass and proportion of the total production of woody tissue browsed by moose. Salix interior plants with experimentally-reduced insect damage produced significantly more stem biomass than controls, but did not differ in stem quality as indicated by nitrogen concentration or protein precipitation capacity, an assay of the protein-binding activity of tannins. CONCLUSIONS: Insect herbivory on Salix, including the outbreak herbivore M. salicifoliella, affected the feeding behavior of moose. The results demonstrate that even moderate levels of leaf damage by insects can have surprisingly strong impacts on stem production and influence the foraging behavior of distantly related taxa browsing on woody tissue months after leaves have dropped.

Longitudinal predictors of self-rated health and mortality in older adults.

INTRODUCTION: Few studies have compared the effects of demographic, cognitive, and behavioral factors of health and mortality longitudinally. We examined predictors of self-rated health and mortality at 3 points, each 2 years apart, over 4 years. METHODS: We used data from the 2006 wave of the Health and Retirement Study and health and mortality indicators from 2006, 2008, and 2010. We analyzed data from 17,930 adults (aged 50-104 y) to examine predictors of self-rated health and data from a subgroup of 1,171 adults who died from 2006 through 2010 to examine predictors of mortality. RESULTS: Time 1 depression was the strongest predictor of self-rated health at all points, independent of age and education. Education, mild activities, body mass index, delayed word recall, and smoking were all associated with self-rated health at each point and predicted mortality. Delayed word recall mediated the relationships of mild activity with health and mortality. Bidirectional mediation was found for the effects of mild activity and depression on health. CONCLUSION: Medical professionals should consider screening for depression and memory difficulties in addition to conducting medical assessments. These assessments could lead to more effective biopsychosocial interventions to help older adults manage risks for mortality.

Parental resource and offspring liability: the influence of extrafloral nectar on oviposition by a leaf-mining moth.

For many insect herbivores, maternal host selection is a critical determinant of offspring survival; however, maternal fitness is also affected by adult resources such as food availability. Consequently, adult resources may promote oviposition in sub-optimal locations when measured in terms of offspring performance. We tested whether oviposition site preference is primarily shaped by proximity to adult food resources or offspring performance in the aspen leaf miner (Phyllocnistis populiella). Quaking aspen (Populus tremuloides) produce extrafloral nectaries (EFNs) on a subset of their leaves. EFN expression on leaves is associated with decreased P. populiella damage and larval performance; however, P. populiella adults feed from EFNs. We reduced extrafloral nectar availability on entire aspen ramets and excluded crawling predators in a full factorial experiment at two sites in interior Alaska, USA. Patterns of egg deposition by P. populiella appeared to be primarily affected by offspring survival rather than adult resource availability. While oviposition was unaffected by nectar availability, adult moths laid fewer eggs on leaves with than without EFNs. By avoiding leaves with EFNs, moths increased offspring survival. Both moths and predators distinguished between leaves with and without EFNs even when nectar and visual cues were obscured, and therefore may respond to chemical cues associated with EFN expression.

Tenascin-X deficiency mimics Ehlers-Danlos syndrome in mice through alteration of collagen deposition.

Tenascin-X is a large extracellular matrix protein of unknown function. Tenascin-X deficiency in humans is associated with Ehlers-Danlos syndrome, a generalized connective tissue disorder resulting from altered metabolism of the fibrillar collagens. Because TNXB is the first Ehlers-Danlos syndrome gene that does not encode a fibrillar collagen or collagen-modifying enzyme, we suggested that tenascin-X might regulate collagen synthesis or deposition. To test this hypothesis, we inactivated Tnxb in mice. Tnxb-/- mice showed progressive skin hyperextensibility, similar to individuals with Ehlers-Danlos syndrome. Biomechanical testing confirmed increased deformability and reduced tensile strength of their skin. The skin of Tnxb-/- mice was histologically normal, but its collagen content was significantly reduced. At the ultrastructural level, collagen fibrils of Tnxb-/- mice were of normal size and shape, but the density of fibrils in their skin was reduced, commensurate with the reduction in collagen content. Studies of cultured dermal fibroblasts showed that although synthesis of collagen I by Tnxb-/- and wildtype cells was similar, Tnxb-/- fibroblasts failed to deposit collagen I into cell-associated matrix. This study confirms a causative role for TNXB in human Ehlers-Danlos syndrome and suggests that tenascin-X is an essential regulator of collagen deposition by dermal fibroblasts.

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit.

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic event, such as a high-impact load acting on articular cartilage, or by joint instability, as occurs with anterior cruciate ligament rupture. There are no effective therapeutics to prevent PTOA currently. Developing a reliable animal model of PTOA is necessary to better understand the mechanisms by which cartilage damage proceeds and to investigate novel treatment strategies to alleviate or prevent the progression of PTOA. This protocol describes an open, drop tower-based rabbit femoral condyle impact model to induce cartilage damage. This model delivered peak loads of 579.1 ± 71.1 N, and peak stresses of 81.9 ± 10.1 MPa with a time-to-peak load of 2.4 ± 0.5 ms. Articular cartilage from impacted medial femoral condyles (MFCs) had higher rates of apoptotic cells (p = 0.0058) and possessed higher Osteoarthritis Research Society International (OARSI) scores of 3.38 ± 1.43 compared to the non-impacted contralateral MFCs (0.56 ± 0.42), and other cartilage surfaces of the impacted knee (p < 0.0001). No differences in OARSI scores were detected among the non-impacted articular surfaces (p > 0.05).

Correlation analysis of cartilage wear with biochemical composition, viscoelastic properties and friction.

Healthy articular cartilage exhibits remarkable resistance to wear, sustaining mechanical loads and relative motion for decades. However, tissues that replace or repair cartilage defects are much less long lasting. Better information on the compositional and material characteristics that contribute to the wear resistance of healthy cartilage could help guide strategies to replace and repair degenerated tissue. The main objective of this study was to assess the relationship between wear of healthy articular cartilage, its biochemical composition, and its viscoelastic material properties. The correlation of these factors with the coefficient of friction during the wear test was also evaluated. Viscoelastic properties of healthy bovine cartilage were determined via stress relaxation indentation. The same specimens underwent an accelerated, in vitro wear test, and the amount of glycosaminoglycans (GAGs) and collagen released during the wear test were considered measures of wear. The frictional response during the wear test was also recorded. The GAG, collagen and water content and the concentration of the enzymatic collagen crosslink pyridinoline were quantified in tissue that was adjacent to each wear test specimen. Finally, correlation analysis was performed to identify potential relationships between wear characteristics of healthy articular cartilage with its composition, viscoelastic material properties and friction. The findings suggest that stiffer cartilage with higher GAG, collagen and water content has a higher wear resistance. Enzymatic collagen crosslinks also enhance the wear resistance of the collagen network. The parameters of wear, composition, and mechanical stiffness of cartilage were all correlated with one another, suggesting that they are interrelated. However, friction was largely independent of these in this study. The results identify characteristics of healthy articular cartilage that contribute to its remarkable wear resistance. These data may be useful for guiding techniques to restore, regenerate, and stabilize cartilage tissue.

Effect of expansion media containing fibroblast growth factor-2 and dexamethasone on the chondrogenic potential of human adipose-derived stromal cells.

hASCs [human ASCs (adipose derived stromal cells)] proliferate more rapidly in the presence of basic FGF-2 (fibroblast growth factor-2) and Dex (dexamethasone). We have examined the effects of expanding hASCs in media containing these two factors on their chondrogenic differentiation potential. Results show that the addition of FGF-2 and Dex to the expansion medium does not remarkably alter the chondrogenic potential of the cells induced by BMP-6 (bone morphogenetic protein-6), based on chondrogenic gene expression, sGAG (sulfated glycosaminoglycan) accumulation and immunohistochemical observation. This is in direct contrast to previously reported promotion of the osteogenic and adipogenic potential of hASCs by these two factors. Therefore, an expansion medium containing FGF-2, with or without Dex, is appropriate for the fast expansion of hASCs without compromising chondrogenic potential.

Computational investigation of fibrin mechanical and damage properties at the interface between native cartilage and implant.

Scaffold-based tissue-engineered constructs as well as cell-free implants offer promising solutions to focal cartilage lesions. However, adequate mechanical stability of these implants in the lesion is required for successful repair. Fibrin is the most common clinically available adhesive for cartilage implant fixation, but fixation quality using fibrin is not well understood. The objectives of this study were to investigate the conditions leading to damage in the fibrin adhesive and to determine which adhesive properties are important in preventing delamination at the interface. An idealized finite element model of the medial compartment of the knee was created, including a circular defect and an osteochondral implant. Damage and failure of fibrin at the interface was represented by a cohesive zone model with coefficients determined from an inverse finite element method and previously published experimental data. Our results demonstrated that fibrin glue alone may not be strong enough to withstand physiologic loads in vivo while fibrin glue combined with chondrocytes more effectively prevents damage at the interface. The results of this study suggest that fibrin fails mainly in shear during off-axis loading and that adhesive materials that are stronger or more compliant than fibrin may be good alternatives due to decreased failure at the interface. The present model may be used to improve design and testing protocols of bioadhesives and give insight into the failure mechanisms of cartilage implant fixation in the knee joint.

Methods to assess in vitro wear of articular cartilage.

New orthopedic implants for focal cartilage defects replace only a portion of the articulating joint and wear against the opposing cartilage surface. The objective of this study was to investigate different methodologies to quantify cartilage wear for future use in screening potential implant materials and finishes. In determining the optimal test parameters, two different cartilage surface geometries were compared: smaller specimens had a flat surface, while larger ones made contact in the center but not at the edge owing to the curvature of the articulating surface. The cartilage wear of the two geometries was compared using three different techniques: the collagen worn from the cartilage specimens was assessed with a modified wear factor, the surface damage was made visible with Indian ink and was quantified, and the change in surface roughness was measured. To interpret the experimental results, maximum shear stresses were evaluated with sliding contact finite element models. Although the modified wear factor was considered to be the most accurate assessment of cartilage wear, surface damage was an effective, inexpensive, and quick technique to evaluate potential implant materials. Flat specimens showed excessive wear at the edges owing to a non-physiologic stress concentration, while the larger specimens wore more uniformly across the surface. These results will be applied to future studies evaluating prospective implant materials.

A Photochemical Crosslinking Approach to Enhance Resistance to Mechanical Wear and Biochemical Degradation of Articular Cartilage.

OBJECTIVE: The objective of this study was to evaluate photochemical crosslinking using Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and light with a wavelength of 670 nm as a potential therapy to strengthen articular cartilage and prevent tissue degradation. DESIGN: Changes in viscoelastic properties with indentation were used to identify 2 crosslinking protocols for further testing. Crosslinked cartilage was subjected to an in vitro, accelerated wear test. The ability of the crosslinked tissue to resist biochemical degradation via collagenase was also measured. To better understand how photochemical crosslinking with CASPc varies through the depth of the tissue, the distribution of photo-initiator and penetration of light through the tissue depth was characterized. Finally, the effect of CASPc on chondrocyte viability and of co-treatment with an antioxidant was evaluated. RESULTS: The equilibrium modulus was the most sensitive viscoelastic measure of crosslinking. Crosslinking decreased both mechanical wear and collagenase digestion compared with control cartilage. These beneficial effects were realized despite the fact that crosslinking appeared to be localized to a region near the articular surface. In addition, chondrocyte viability was maintained in crosslinked tissue treated with antioxidants. CONCLUSION: These results suggest that photochemical crosslinking with CASPc and 670 nm light holds promise as a potential therapy to prevent cartilage degeneration by protecting cartilage from mechanical wear and biochemical degradation. Limitations were also evident, however, as an antioxidant treatment was necessary to maintain chondrocyte viability in crosslinked tissue.

Defensive effects of extrafloral nectaries in quaking aspen differ with scale.

The effects of plant defenses on herbivory can differ among spatial scales. This may be particularly common with indirect defenses, such as extrafloral nectaries (EFNs), that attract predatory arthropods and are dependent on predator distribution, abundance, and behavior. We tested the defensive effects of EFNs in quaking aspen (Populus tremuloides Michx.) against damage by a specialist herbivore, the aspen leaf miner (Phyllocnistis populiella Cham.), at the scale of individual leaves and entire ramets (i.e., stems). Experiments excluding crawling arthropods revealed that the effects of aspen EFNs differed at the leaf and ramet scales. Crawling predators caused similar reductions in the percent leaf area mined on individual leaves with and without EFNs. However, the extent to which crawling predators increased leaf miner mortality and, consequently, reduced mining damage increased with EFN expression at the ramet scale. Thus, aspen EFNs provided a diffuse defense, reducing damage to leaves across a ramet regardless of leaf-scale EFN expression. We detected lower leaf miner damage and survival unassociated with crawling predators on EFN-bearing leaves, suggesting that direct defenses (e.g., chemical defenses) were stronger on leaves with than without EFNs. Greater direct defenses on EFN-bearing leaves may reduce the probability of losing these leaves and thus weakening ramet-scale EFN defense. Aspen growth was not related to EFN expression or the presence of crawling predators over the course of a single season. Different effects of aspen EFNs at the leaf and ramet scales suggest that future studies may benefit from examining indirect defenses simultaneously at multiple scales.

Bone morphogenetic protein 6 drives both osteogenesis and chondrogenesis in murine adipose-derived mesenchymal cells depending on culture conditions.

Bone morphogenetic proteins (BMPs) play a dual role as a factor in both bone and cartilage development and correspondingly have the therapeutic potential to regenerate both tissues. Given this dual nature, previous in vitro research using BMPs has relied on distinct media formulations and culture conditions to drive undifferentiated cells to the osteogenic or chondrogenic lineage. To isolate the impact of culture conditions and to explore the effect of BMP-6 on murine adipose-derived mesenchymal cells (ASCs), ASCs were seeded in either monolayer or pellets in an identical medium containing BMP-6. Results indicate that BMP-6 differentially promotes osteogenesis and chondrogenesis in ASCs depending on culture conditions. BMP-6 potently induced alkaline phosphatase activity and mineralization in ASCs cultured in monolayer conditions. In contrast, BMP-6 enhanced proteoglycan accumulation in ASCs seeded in chondrogenic pellet culture. A comparison of gene expression suggests that the differentiating effect of BMP-6 is specific to the particular culture condition. This study highlights the importance of the interactions between chemical signaling and microenvironmental cues in directing cell fate.

Induction of phenolic glycosides by quaking aspen (Populus tremuloides) leaves in relation to extrafloral nectaries and epidermal leaf mining.

We studied the effect of epidermal leaf mining on the leaf chemistry of quaking aspen, Populus tremuloides, during an outbreak of the aspen leaf miner, Phyllocnistis populiella, in the boreal forest of interior Alaska. Phyllocnistis populiella feeds on the epidermal cells of P. tremuloides leaves. Eleven days after the onset of leaf mining, concentrations of the phenolic glycosides tremulacin and salicortin were significantly higher in aspen leaves that had received natural levels of leaf mining than in leaves sprayed with insecticide to reduce mining damage. In a second experiment, we examined the time course of induction in more detail. The levels of foliar phenolic glycosides in naturally mined ramets increased relative to the levels in insecticide-treated ramets on the ninth day following the onset of leaf mining. Induction occurred while some leaf miner larvae were still feeding and when leaves had sustained mining over 5% of the leaf surface. Leaves with extrafloral nectaries (EFNs) had significantly higher constitutive and induced levels of phenolic glycosides than leaves lacking EFNs, but there was no difference in the ability of leaves with and without EFNs to induce phenolic glycosides in response to mining. Previous work showed that the extent of leaf mining damage was negatively related to the total foliar phenolic glycoside concentration, suggesting that phenolic glycosides deter or reduce mining damage. The results presented here demonstrate that induction of phenolic glycosides can be triggered by relatively small amounts of mining damage confined to the epidermal tissue, and that these changes in leaf chemistry occur while a subset of leaf miners are still feeding within the leaf.

The leaf miner Phyllocnistis populiella negatively impacts water relations in aspen.

Within the North American boreal forest, a widespread outbreak of the epidermal leaf miner Phyllocnistis populiella Cham. has damaged quaking aspen (Populus tremuloides Michx.) for nearly 20 years. In a series of experiments, we tested the effects of feeding damage by P. populiella on leaf water relations and gas exchange. Relative to insecticide-treated trees, the leaves of naturally mined trees had lower photosynthesis, stomatal conductance to water vapor, transpiration, water-use efficiency, predawn water potential and water content, as well as more enriched foliar δ13C. The magnitude of the difference between naturally mined and insecticide-treated trees did not change significantly throughout the growing season, suggesting that the effect is not caused by accumulation of incidental damage to mined portions of the epidermis over time. The contributions of mining-related stomatal malfunction and cuticular transpiration to these overall effects were investigated by restricting mining damage to stomatous abaxial and astomatous adaxial leaf surfaces. Mining of the abaxial epidermis decreased photosynthesis and enriched leaf δ13C, while increasing leaf water potential and water content relative to unmined leaves, effects consistent with stomatal closure due to disfunction of mined guard cells. Mining of the adaxial epidermis also reduced photosynthesis but had different effects on water relations, reducing midday leaf water potential and water content relative to unmined leaves, and did not affect δ13C. In the laboratory, extent of mining damage to the adaxial surface was positively related to the rate of water loss by leaves treated to prevent water loss through stomata. We conclude that overall, despite water savings due to closure of mined stomata, natural levels of damage by P. populiella negatively impact water relations due to increased cuticular permeability to water vapor across the mined portions of the epidermis. Leaf mining by P. populiella could exacerbate the negative effects of climate warming and water deficit in interior Alaska.

Anisotropic properties of articular cartilage in an accelerated in vitro wear test.

Many material properties of articular cartilage are anisotropic, particularly in the superficial zone where collagen fibers have a preferential direction. However, the anisotropy of cartilage wear had not been previously investigated. The objective of this study was to evaluate the anisotropy of cartilage material behavior in an in vitro wear test. The wear and coefficient of friction of bovine condylar cartilage were measured with loading in directions parallel (longitudinal) and orthogonal (transverse) to the collagen fiber orientation at the articular surface. An accelerated cartilage wear test was performed against a T316 stainless-steel plate in a solution of phosphate buffered saline with protease inhibitors. A constant load of 160 N was maintained for 14000 cycles of reciprocal sliding motion at 4 mm/s velocity and a travel distance of 18 mm in each direction. The contact pressure during the wear test was approximately 2 MPa, which is in the range of that reported in the human knee and hip joint. Wear was measured by biochemically quantifying the glycosaminoglycans (GAGs) and collagen that was released from the tissue during the wear test. Collagen damage was evaluated with collagen hybridizing peptide (CHP), while visualization of the tissue composition after the wear test was provided with histologic analysis. Results demonstrated that wear in the transverse direction released about twice as many GAGs than in the longitudinal direction, but that no significant differences were seen in the amount of collagen released from the specimens. Specimens worn in the transverse direction had a higher intensity of CHP stain than those worn in the longitudinal direction, suggesting more collagen damage from wear in the transverse direction. No anisotropy in friction was detected at any point in the wear test. Histologic and CHP images demonstrate that the GAG loss and collagen damage extended through much of the depth of the cartilage tissue, particularly for wear in the transverse direction. These results highlight distinct differences between cartilage wear and the wear of traditional engineering materials, and suggest that further study on cartilage wear is warranted. A potential clinical implication of these results is that orienting osteochondral grafts such that the direction of wear is aligned with the primary fiber direction at the articular surface may optimize the life of the graft.

Mineral deposition and vascular invasion of hydroxyapatite reinforced collagen scaffolds seeded with human adipose-derived stem cells.

BACKGROUND: Collagen-based scaffolds reinforced with hydroxyapatite (HA) are an attractive choice for bone tissue engineering because their composition mimics that of bone. We previously reported the development of compression-molded collagen-HA scaffolds that exhibited high porosity, interconnected pores, and mechanical properties that were well-suited for surgical handling and fixation. The objective of this study was to investigate these novel collagen-HA scaffolds in combination with human adipose-derived stem cells (hASCs) as a template for bone formation in a subcutaneous athymic mouse model. METHODS: Collagen-HA scaffolds and collagen-only scaffolds were fabricated as previously described, and a clinically approved bone void filler was used as a control for the material. Constructs were seeded with hASCs and were pre-treated with either control or osteogenic media. A cell-free group was also included. Scaffolds were implanted subcutaneously in the backs of athymic nude mice for 8 weeks. Mineral deposition was quantified via micro-computed tomography. Histological and immunofluorescence images of the explants were used to analyze their vascular invasion, remodeling and cellularity. RESULTS: Cell-free collagen-HA scaffolds and those that were pre-seeded with osteogenically differentiated hASCs supported mineral deposition and vascular invasion at comparable rates, while cell-seeded constructs treated with the control medium showed lower mineralization after implantation. HA-reinforcement allowed collagen constructs to maintain their shape, provided improved cell-tissue-scaffold integration, and resulted in a more organized tissue when pre-treated in an osteogenic medium. Scaffold type and pre-treatment also determined osteoclast activity and therefore potential remodeling of the constructs. CONCLUSIONS: The results of this study cumulatively indicate that treatment medium and scaffold composition direct mineralization and angiogenic tissue formation in an ectopic model. The data suggest that it may be necessary to match the scaffold with a particular cell type and cell-specific pre-treatment to achieve optimal bone formation.

Scaffold-free Bioprinting of Mesenchymal Stem Cells with the Regenova Printer: Optimization of Printing Parameters.

The Kenzan bioprinting method provides a high-resolution biofabrication process by facilitating the fusion of submillimeter cell aggregates (spheroids) into larger tissue constructs on a needle array that is removed upon spheroid fusion. Although the method is relatively straightforward in principle, Kenzan method bioprinting relies on a complex 3D bioprinter (Regenova Bio 3D Printer, Cyfuse, K.K., Japan) implementing an advanced vision system to verify the microscopic spheroids' geometry and high-precision mechatronics to aseptically manipulate the spheroids into position. Due to the complexity of the operation, the need for aseptic conditions, and the size of the spheroids, proficiency with the Regenova Bio 3D Printer and the Kenzan method requires development of best practices and troubleshooting techniques to ensure a robust print and minimize the use of resources. In addition, managing the construct post-bioprinting both in culture and for surgical implantation requires careful consideration and workflow design. Here, we describe methods for generating a competent tissue construct and optimizing the bioprinting process. Optimization resulted in a 4-fold reduction in print times, a 20-fold reduction in the use of bioprinting nozzles, and more robust constructs. The results and procedures described herein will have potential applications for tissue engineering, research, and clinical uses in the future.

Scaffold-free bioprinting of mesenchymal stem cells using the Regenova printer: Spheroid characterization and osteogenic differentiation.

Limitations in scaffold material properties, such as sub-optimal degradation time, highlight the need for alternative approaches to engineer de novo tissues. One emerging solution for fabricating tissue constructs is scaffold-free tissue engineering. To facilitate this approach, three-dimensional (3D) bioprinting technology (Regenova Bio 3D Printer) has been developed to construct complex geometric shapes from discrete cellular spheroids without exogenous scaffolds. Optimizing spheroid fabrication and characterizing cellular behavior in the spheroid environment are important first steps prior to printing larger constructs. Here, we characterized spheroids of immortalized mouse bone marrow stromal cells (BMSCs) that were differentiated to the osteogenic lineage. Immortalized BMSCs were seeded in low attachment 96-well plates in various numbers to generate self-aggregated spheroids either under the force of gravity or centrifugation. Cells were cultured in control or osteogenic media for up to 28 days. Spheroid diameter, roundness and smoothness were measured. Cell viability, DNA content and alkaline phosphatase activity were assessed at multiple time points. Additionally, expression of osteogenic markers was determined using real time qPCR. Spheroids formed under gravity with 20 K, 30 K and 40 K cells had average diameters of 498.5 ± 8.3 µm, 580.0 ± 32.9 µm and 639.2 ± 54.0 µm, respectively, while those formed under 300G centrifugation with the same numbers of cells had average diameters of 362.3 ± 3.5 µm, 433.1 ± 6.4 µm and 491.2 ± 8.0 µm. Spheroids formed via centrifugation were superior to those formed by gravity, as evidenced by better roundness and smoothness and double the retention of DNA (cellular) content. Cells in spheroids exhibited a robust osteogenic response to the differentiation medium, including higher mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than those cultured in control medium, as well as greater alkaline phosphatase activity. The optimal spheroid fabrication technique from this study was to aggregate 40K cells under 150-300G centrifugation. In future investigations, these spheroids will be 3D printed into larger tissue constructs.

Dysfunctional stem and progenitor cells impair fracture healing with age.

Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells (MSCs) are directed to replace the bone tissue, while endothelial progenitor cells (EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species, and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics. Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly.