Optimizing Cartilage Tissue Engineering through Computational Growth Models and Experimental Culture Protocols

Osteoarthritis is a debilitating and irreversible disease afflicting the synovial joints. It is characterized by pain and hindered mobility. Given that osteoarthritis has no cure, current treatments focus on pain management. Ultimately, however, a patient's pain and immobility necessitates joint replacement surgery. An attractive alternative to this treatment paradigm, tissue engineering is a promising strategy for resurfacing the osteoarthritis-afflicted cartilage surface with a biochemically and biomechanically similar tissue to the healthy native cartilage tissue. The most successful cartilage tissue engineered systems to date can repeatably grow constructs ~4 mm in diameter with native proteoglycan and compressive mechanical properties. Unfortunately, as symptomatic cartilage typically presents only once lesions span large regions of the joint (~25 mm in diameter), these small construct are of limited use in clinical practice. Numerous attempts to simply grow a construct large enough to span the size of an osteoarthritic lesion have shown that the growth of large engineered tissues develop heterogeneous properties, emphasizing the need for culture protocols to enhance tissue homogeneity and robustness. In particular, as nutrient limitations drive heterogeneous growth in engineered cartilage, developing strategies to improve nutrition throughout the construct are critical for clinical translation of the technology. To this end, our lab has successfully supplemented nutrient channels within large engineered cartilage constructs to improve the functional properties of developing tissue. However, it is unknown what the optimal nutrient channel spacing is for growing large cartilage constructs of anatomical scale. Additionally, the fundamental factors and mechanisms which drive tissue heterogeneity have not been implicated, making the results of channel-spacing optimizations difficult to translate across different systems. Computational models of growth, faithful to the physics and biology of engineered tissue growth, may serve as an insightful and efficient tool for optimally designing culture protocols and construct geometries to ensure homogeneous matrix deposition. Such computational tools, however, are not presently available, owing to the unresolved mechanical and biological growth phenomena within developing engineered cartilage. This dissertation seeks to develop and implement computational models for predicting the biochemical and biomechanical growth of engineered tissues and apply these models to optimizing tissue culture strategies. These models are developed in two stages: 1) based on our recent characterization of the nutrient demands of engineered cartilage, models are developed to simulate the spatial biochemical deposition of matrix within tissue constructs and, subsequently, 2) based on models of biochemical matrix deposition we develop models for simulating the mechanical growth of tissue constructs. To accomplish these tasks, we first develop models simulating glucose availability within large tissue constructs using system-specific modeling based on our recent characterization of the glucose demands of engineered cartilage. These models led to early insight that we had to enhance the supply of glucose within large tissue constructs to ensure maximal matrix synthesis throughout culture. Experimental validations confirmed that increasing glucose supply enhanced matrix deposition and growth in large tissue constructs. However, even despite the increased glucose supply, increasing the size of constructs demonstrated that severe matrix heterogeneities were still present. Subsequent nutrient characterization led to the finding that TGF-ß transport was significantly hindered within large tissue constructs. Incorporating the influence of glucose and TGF-ß into the computational model growth kinetics. Using both nutrients, models recreated the heterogeneous matrix deposition evident in our earlier work and could account for the role of cell seeding density and construct geometry on tissue growth. The insights gathered from this modeling analysis led to important changes in our culture protocols: we could reduce the dose of TGF-ß from 10 ng/mL to 1 ng/mL for constructs cultured with channels, saving considerable expense while still maintaining a high level of matrix synthesis throughout the construct. In the presence of sufficient nutrition, we witnessed an unprecedented level of matrix deposition and physical growth of the constructs. In fact, by using developmentally physiologic cell seeding densities (120 million cells/mL) and providing adequate nutrition, constructs physically grew to 9-times their originally cast size. Despite such encouraging growth, tissue function properties plateaued at sub-physiologic levels. For insight into the connection between matrix deposition and tissue mechanics, we extended the computational growth models to consider the mechanisms underlying physical growth. Interestingly, we found that a large matrix synthesis mismatch between proteoglycans and collagen gave rise to the excessive tissue swelling. Computational models of this matrix synthesis mismatch predicted the high tissue swelling displayed experimentally only when a damage-able collagen fiber material was implemented. Together, the experimental and modeling evidence suggested a new mechanism of cartilage growth: the high proteoglycan deposition creates a swelling pressure within the nascent tissue which outcompetes the restraining force of newly deposited collagens; this rapid tissue swelling disrupts a functional collagen network from forming. Subsequent analysis suggested that the disruption of the collagen network prevented the formation of collagen crosslinks, stymieing the development of native functional properties. Based on this insight into the mechanisms of cartilage growth, we developed a culture systems to improve tissue functional properties. Modeling analysis indicated two novel routes for improving tissue mechanics: either through 1) reducing the swelling response (synthesis and deposition) of proteoglycans or 2) enhancing and reinforcing the newly synthesized collagen to prevent disruptions brought on by tissue swelling. We developed a cage culture system for resisting the swelling pressure of deposited proteoglycans and reenforcing the deposition of new collagens. Using this cage system, we grew tissue constructs with enhanced functional properties using two separate scaffold systems – agarose and a cartilage-derived matrix hydrogel – suggesting this mechanism of growth is fundamental to engineered cartilage development. This work has generated a novel paradigm towards engineering cartilage constructs using biomimetic strategies. Performing simulations with the validated, computational growth models allowed anatomically-sized cartilage constructs to grow into the largest, homogeneous cartilage constructs grown to date. Models presented a new level of insight into the nutrient demands of developing tissues, allowing for the first time the successful development of large tissue constructs grown with developmentally physiologic cell seeding densities. In this way, tissue constructs growth followed a biomimetic approach, based on the high cell densities and cartilage canals and vasculature present during fetal cartilage development. Adequate nutrition led to high levels of tissue growth not previously experienced in vitro, a result of adequately nourishing primary chondrocytes, a cell type which preferentially deposits proteoglycans over collagen. We therefore developed a cage-based growth system to resist the proteoglycan-induced tissue swelling in a manner similar to the fetal development of cartilage where the resident cells synthesize more collagens than proteoglycans. Together, the use of nutrient growth models, high cell seeding densities, and culture systems to strengthen the collagen-framework of de novo cartilage proved beneficial for engineering anatomically-sized cartilage constructs. The fundamental mechanisms identified here are likely to be universal across a number of engineered cartilage systems and will be adapted to more clinically-relevant cell sources in future our work

Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

The primary goal of this study was to characterize the response of chondrocyte-seeded agarose constructs to varying concentrations of several key nutrients in a chondrogenic medium, within the overall context of optimizing the key nutrients and the placement of nutrient channels for successful growth of cartilage tissue constructs large enough to be clinically relevant in the treatment of osteoarthritis (OA). To this end, chondrocyte-agarose constructs (phi4x2.34 mm, 30x106 cells/mL) were subjected to varying supplementation levels of insulin (0× to 30× relative to standard supplementation), transferrin (0x to 30x), selenous acid (0x to 10x), ascorbate (0x to 30x), and glucose (0x to 3x). The quality of resulting engineered tissue constructs was evaluated by their compressive modulus (E-Y), tensile modulus (E+Y), hydraulic permeability (k), and content of sulfated glycosaminoglycans (sGAG) and collagen (COL); DNA content was also quantified. Three control groups from two separate castings of constructs (1x concentrations of all medium constituents) were used. After 42 days of culture, values in each of these controls were, respectively, E-Y=518 plus or minus 78, 401 plus or minus 113, 236 plus or minus 67 kPa; E+Y=1420 plus or minus 430, 1140 plus or minus 490, 1240 plus or minus 280 kPa; k=2.3 plus or minus 0.8x10-3, 5.4 plus or minus 7.0x10-3, 3.3 plus or minus 1.3x10-3 mm4/N times s; sGAG=7.8 plus or minus 0.3, 6.3 plus or minus 0.4, 4.1 plus or minus 0.5%/ww; COL=1.3 plus or minus 0.2, 1.1 plus or minus 0.3, 1.4 plus or minus 0.4%/ww; and DNA=11.5 plus or minus 2.2, 12.1 plus or minus 0.6, 5.2 plus or minus 2.8 μg/disk. The presence of insulin and ascorbate was essential, but their concentrations may drop as low as 0.3x without detrimental effects on any of the measured properties; excessive supplementation of ascorbate (up to 30x) was detrimental to E-Y, and 30x insulin was detrimental to both E+Y and E-Y. The presence of glucose was similarly essential, and matrix elaboration was significantly dependent on its concentration (p less than 10-6), with loss of functional properties, composition, and cellularity observed at less than or equal to 0.3x; excessive glucose supplementation (up to 3x) showed no detrimental effects. In contrast, transferrin and selenous acid had no influence on matrix elaboration. These findings suggest that adequate distributions of insulin, ascorbate, and glucose, but not necessarily of transferrin and selenous acid, must be ensured within large engineered cartilage constructs to produce a viable substitute for joint tissue lost due to OA

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues

An environmental (pre)history of European fishing: past and future archaeological contributions to sustainable fisheries.

This paper explores the past and potential contribution of archaeology to marine historical ecology. The primary focus is European fishing of marine and diadromous taxa, with global comparisons highlighting the wider applicability of archaeological approaches. The review illustrates how study of excavated fish bones, otoliths and shells can inform our understanding of: (a) changes in biogeography, including the previous distribution of lost species; (b) long-term fluctuations in the aquatic environment, including climate change; (c) the intensity of exploitation and other anthropogenic effects; (d) trade, commodification and globalisation. These issues are also relevant to inform fisheries conservation and management targets. Equally important, the long (pre)history of European fishing raises awareness of our ecological heritage debt, owed for centuries of wealth, sustenance and well-being, and for which we share collective responsibility. This debt represents both a loss and a reason for optimism, insofar as it is a reservoir of potential to be filled by careful stewardship of our rivers, lakes, seas and oceans

Resection of the Liver for Colorectal Carcinoma Metastases A Multi-institutional Study of Long-term Survivors

In this review of a collected series of patients undergoing hepatic resection for colorectal metastases, 100 patients were found to have survived greater than five years from the time of resection. Of these 100 long-term survivors, 71 remain disease-free through the last follow-up, 19 recurred prior to five years, and ten recurred after five years. Patient characteristics that may have contributed to survival were examined. Procedures performed included five trisegmentectomies, 32 lobectomies, 16 left lateral segmentectomies, and 45 wedge resections. The margin of resection was recorded in 27 patients, one of whom had a positive margin, nine of whom had a less than or equal to l-cm margin, and 17 of whom had a greater than 1-cm margin. Eighty-one patients had a solitary metastasis to the liver, 11 patients had two metastases, one patient had three metastases, and four patients had four metastases. Thirty patients had Stage C primary carcinoma, 40 had Stage B primary carcinoma, and one had Stage A primary carcinoma. The disease-free interval from the time of colon resection to the time of liver resection was less than one year in 65 patients, and greater than one year in 34 patients. Three patients had bilobar metastases. Four of the patients had extrahepatic disease resected simultaneously with the liver resection. Though several contraindications to hepatic resection have been proposed in the past, five-year survival has been found in patients with extrahepatic disease resected simultaneously, patients with bilobar metastases, patients with multiple metastases, and patients with positive margins. Five-year disease-free survivors are also present in each of these subsets. It is concluded that five-year survival is possible in the presence of reported contraindications to resection, and therefore that the decision to resect the liver must be individualized

Western wind : an introduction to poetry

Western Wind continues in its forth edition to provide brilliant eludication of the elements of poetry, as well as an outstanding collection of classic and contemporary poems.xxxviii, 631 p.: ill.; 23 c

SHARP-LINE LUMINESCENCE OF THE HEXABROMORHENATE (IV) ION AND THE HEXABROMOOSMATE (IV) ION IN CESIUM HEXABROMOZIRCONATE (IV) AT 20∘20^{\circ} K

Author Institution: Chemistry Department, University of MaineStrong sharp-line luminescence has been observed for $Re^{+4} (5d^{3})$ and $Os^{+4} (5d^{4})$ in single cubic crystals of the host lattice $Cs_{2}ZrBr_{6}$ at $20^{\circ} K$. For $Re^{+4}$, luminescence and absorption spectra are reported within the $t^{3}_{2g}$ configuration between the ${\Gamma}_{7}(^{2}T_{2g})$ and ${\Gamma}_{8}(^{2}T_{2g})$ states and the ${\Gamma}_{8}(^{4}A_{2g})$ ground state. Calculated vibrational mode energies of the ground electronic state are found to be in good agreement with the available infrared and Raman data for the $ReBr^{-2}_{6}$ complex and to be somewhat different than the vibrational mode energies for the ${\Gamma}_{7} (^{2}T_{2g})$ and ${\Gamma}_{8}(^{2}T_{2g})$ excited electronic states. For the electronic-dipole portion of the ${\Gamma}_{8}(^{4}A_{2g}) \rightarrow {\Gamma}_{7}(^{2}T_{2g})$ transition short progressions are present not only in the $a_{1g}$ symmetric mode but also in the $e_{g}$ and $t_{2g}$ modes. Similar results have been obtained for the $OsBr^{-2}_{6}$ complex