Development of a whole organ culture model for intervertebral disc disease.

Background/objectiveWhole organ in vitro intervertebral disc models have been associated with poor maintenance of cell viability. No previous studies have used a rotating wall vessel bioreactor for intervertebral disc explants culture. The purpose of this study was to develop and validate an in vitro model for the assessment of biological and biomechanical measures of intervertebral disc health and disease.MethodsTo this end, endplate-intervertebral disc-endplate whole organ explants were harvested from the tails of rats. For the injured group, the annulus fibrosus was penetrated with a 20G needle to the nucleus pulposus and aspirated. Explants were cultured in a rotating wall vessel bioreactor for 14 days.ResultsCell viability and histologic assessments were performed at Day 0, Day 1, Day 7, and Day 14. Compressive mechanical properties of the intervertebral disc were assessed at Day 0 and Day 14. In the annulus fibrosus and nucleus pulposus cells, the uninjured group maintained high viability through 14 days of culture, whereas cell viability in annulus fibrosus and nucleus pulposus of the injured intervertebral discs was markedly lower at Day 7 and Day 14. Histologically, the uninjured intervertebral discs maintained cell viability and tissue morphology and architecture through 14 days, whereas the injured intervertebral discs showed areas of cell death, loss of extracellular matrix integrity, and architecture by Day 14. Stiffness values for uninjured intervertebral discs were similar at Day 0 and Day 14, whereas the stiffness for the injured intervertebral discs was approximately 2.5 times greater at Day 14.ConclusionThese results suggest that whole organ intervertebral discs explants can be successfully cultured in a rotating wall vessel bioreactor to maintain cell viability and tissue architecture in both annulus fibrosus and nucleus pulposus for at least 14 days. In addition, the injury used produced pathologic changes consistent with those seen in degenerative intervertebral disc disease in humans. This model will permit further study into potential future treatments and other mechanisms of addressing intervertebral disc disease

Development of a whole organ culture model for intervertebral disc disease

Background/Objective: Whole organ in vitro intervertebral disc models have been associated with poor maintenance of cell viability. No previous studies have used a rotating wall vessel bioreactor for intervertebral disc explants culture. The purpose of this study was to develop and validate an in vitro model for the assessment of biological and biomechanical measures of intervertebral disc health and disease. Methods: To this end, endplate-intervertebral disc-endplate whole organ explants were harvested from the tails of rats. For the injured group, the annulus fibrosus was penetrated with a 20G needle to the nucleus pulposus and aspirated. Explants were cultured in a rotating wall vessel bioreactor for 14 days. Results: Cell viability and histologic assessments were performed at Day 0, Day 1, Day 7, and Day 14. Compressive mechanical properties of the intervertebral disc were assessed at Day 0 and Day 14. In the annulus fibrosus and nucleus pulposus cells, the uninjured group maintained high viability through 14 days of culture, whereas cell viability in annulus fibrosus and nucleus pulposus of the injured intervertebral discs was markedly lower at Day 7 and Day 14. Histologically, the uninjured intervertebral discs maintained cell viability and tissue morphology and architecture through 14 days, whereas the injured intervertebral discs showed areas of cell death, loss of extracellular matrix integrity, and architecture by Day 14. Stiffness values for uninjured intervertebral discs were similar at Day 0 and Day 14, whereas the stiffness for the injured intervertebral discs was approximately 2.5 times greater at Day 14. Conclusion: These results suggest that whole organ intervertebral discs explants can be successfully cultured in a rotating wall vessel bioreactor to maintain cell viability and tissue architecture in both annulus fibrosus and nucleus pulposus for at least 14 days. In addition, the injury used produced pathologic changes consistent with those seen in degenerative intervertebral disc disease in humans. This model will permit further study into potential future treatments and other mechanisms of addressing intervertebral disc disease

Validation of a low-cost, carbon dioxide-based cryoablation system for percutaneous tumor ablation.

Breast cancer rates are rising in low- and middle-income countries (LMICs), yet there is a lack of accessible and cost-effective treatment. As a result, the cancer burden and death rates are highest in LMICs. In an effort to meet this need, our work presents the design and feasibility of a low-cost cryoablation system using widely-available carbon dioxide as the only consumable. This system uses an 8-gauge outer-diameter needle and Joule-Thomson expansion to percutaneously necrose tissue with cryoablation. Bench top experiments characterized temperature dynamics in ultrasound gel demonstrated that isotherms greater than 2 cm were formed. Further, this system was applied to mammary tumors in an in vivo rat model and necrosis was verified by histopathology. Finally, freezing capacity under a large heat load was assessed with an in vivo porcine study, where volumes of necrosis greater than 1.5 cm in diameter confirmed by histopathology were induced in a highly perfused liver after two 7-minute freeze cycles. These results demonstrate the feasibility of a carbon-dioxide based cryoablation system for improving solid tumor treatment options in resource-constrained environments

Investigation of the Microheterogeneity and Aglycone Specificity-Conferring Residues of Black Cherry Prunasin Hydrolases

In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase. Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase. Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members. Their respective cDNAs (designated Ph1, Ph2, Ph3, Ph4, and Ph5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs. Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites. Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns. Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates. Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris. Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis. The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity

Ecological Interface for Assessing Cardiac Disease

Using the ecological interface design approach, a graphical user interface was developed to show how different factors (e.g., LDL, HDL, triglycerides, systolic blood pressure) contribute to cardiac health. The display is based on an epidemiological model derived from the Framingham study and additional treatment guidelines in the medical literature. This interactive display allows physicians and patients to see how different factors contribute to overall cardiac health and to see the impact of interventions on reducing risk. The display also graphically associates the state of the patient with treatment categories to help physicians to select the best treatment method based on empirical models. It also has the potential to enrich the dialogue between physician and patient through interactive ‘experiments’ that illustrate the potential benefits of various treatment options