Ongoing Research at Flexcell®
Below are abstracts from recently presented or published research projects onging at Flexcell® or conducted in collaboration with Flexcell®.

Out of Academics: Education, Entrepreneurship and Enterprise
Banes AJ. Ann Biomed Eng. 2013 Jun 25. [Epub ahead of print]

The author started a niche biotech company in 1985 called Flexcell® to distribute an enabling technology, mechanobiology devices, to the field. He was the first University of North Carolina faculty member to start a company and stay with it as he pursued his career in academics. That was an unpopular route at that time, but a path he was driven to navigate. Those interests, merged with his training, led to the design and manufacture of mechanobiology devices such as the Flexercell® Strain Unit and the BioFlex® flexible bottom culture plates to study fundamental responses of cells to strain. Principles in these devices were also incorporated into bioreactors for tissue engineering, which are standard in the marketplace today. In this article, the major roadblocks will be chronicled that were overcome to help build the field of mechanobiology and create a small biotechnology company. Through example, the author's formula for achieving milestones will be discussed including, the DRIVE it takes to get there ["DRIVE": Determination (Confidence), Research and Development (R&D) and Risk-Taking, Innovation (Imagination) and Intellectual Property, achieving Victory, and Enterprise.

Differential expression and cellular localization of novel isoforms of the tendon biomarker tenomodulin
Qi J, Dmochowski JM, Banes AN, Tsuzaki M, Bynum D, Patterson M, Creighton A, Gomez S, Tech K, Cederlund A, Banes AJ. J Appl Physiol 113(6):861-71, 2012. doi: 10.1152/japplphysiol.00198.2012. Epub 2012 Jun 14.

Tenomodulin (Tnmd, also called Tendin) is classified as a type II transmembrane glycoprotein and is highly expressed in developing as well as in mature tendons. Along with scleraxis (scx), Tnmd is a candidate marker gene for tenocytes. Its function is unknown, but it has been reported to have anti-angiogenic properties. Results in a knockout mouse model did not substantiate that claim. It has homology to chondromodulin-I. Single nucleotide polymorphisms of TNMD have been associated with obesity, macular degeneration, and Alzheimer's disease in patients. In the present study, three Tnmd isoforms with deduced molecular weights of 20.3 (isoform II), 25.4 (isoform III), and 37.1 (isoform I) kDa were proposed and verified by Western blot from cells with green fluorescent protein-linked, overexpressed constructs, tissue, and by qPCR of isoforms from human tissues and cultured cells. Overexpression of each Tnmd isoform followed by immunofluorescence imaging showed that isoforms I and II had perinuclear localization while isoform III was cytoplasmic. Results of qPCR demonstrated differential expression of each Tnmd isoform in patient's specimens taken from flexor carpi radialis, biceps brachii, and flexor digitorum profundus tendons. Knockdown of Tnmd increased the expression of both scleraxis (scx) and myostatin, indicating a potential negative feedback loop between Tnmd and its regulators. Knockdown of all Tnmd isoforms simultaneously also reduced tenocyte proliferation. I-TASSER protein three-dimensional conformation modeling predictions indicated each Tnmd isoform had different structures and potential functions: isoform 1, modeled as a cytosine methyltransferase; isoform 2, a SUMO-1-like SENP-1 protease; and isoform 3, an a-syntrophin, plextrin homology domain scaffolding protein. Further functional studies with each Tnmd isoform may help us to better understand regulation of tenocyte proliferation, tendon development, response to injury and strain, as well as mechanisms in tendinoses. These results may indicate novel therapeutic targets in specific tenomodulin isoforms as well as treatments for tendon diseases.

Collagel® and Thermacol®: Collagen Hydrogel Formulations for Tissue Engineering Applications
Banes AJ, Amegashie A, McGee C. Regenerative Medicine Symposium, Research Triangle Park, NC, October 15, 2013.

Introduction: Three dimensional matrices for use in cell culture applications began with the use of fibrin clots in organ culture and acid-soluble rat tail tendon collagen adsorption to glass slides. Two early commercially available products for use in the 3D cell culture market included Matrigel™, a matrix isolated from Englebreth Holm Swarm chondrosarcoma cells, and Vitrogen® (now Purecol®, Advanced Biomatrix), an atelopeptide type I bovine collagen in solution. Collagel® is a formulation of porcine Achilles tendon (ATs) collagen with long fibrils in vitro. Thermacol® is a specially formulated mixture of collagen hydrogels of telopeptide and atelopeptide collagens isolated form porcine ATs (patent pending). Advantages of the novel formulation include longer collagen fibrils and faster, thermally sensitive gelation times. Methods: Type I collagen was isolated from porcine ATs or bovine dermis after morcelization of the cleaned ATs, pulverization and acid extractions, followed by chromatographic clean-up. Results: A formulation of telopeptide-containing collagen from tendon combined with atelopeptide collagen from dermis resulted in controlled thermal gelation when hydrogels were shifted to 37░C. Collagen fibrils in the porcine Collagel« hydrogels were significantly longer than those that comprised bovine dermis collagen preparations as evidenced by reflection interference microscopy. The thermally sensitive Thermacol® preparation in 10/90 or 20/80 telo to atelo-peptide formulations yielded a hydrogel that rapidly gelled when shifted from room temperature to 37░C. Conclusions: Mixtures of atelo and telo-peptide-containing collagen hydrogels show superior gelation kinetics to bovine dermis-derived collagens for use in cell-gel constructs for tissue engineering applications.

Comparative Matrix Compaction and Cell Survival Analysis of Linear 3D Bioartificial Tissues
McGee CE, Amegashie A, Brown F, Grant D, Banes A, Banes AJ. Regenerative Medicine Symposium, Research Triangle Park, NC, October 15, 2013.

Introduction: The Tissue Train® culture systems facilitate the in vitro growth of cultured primary and continuous cells in a three-dimensional (3D) collagen matrix thereby subjecting them to more native growth conditions with the potential for applying static or cyclic mechanical loading. Bioartificial tissues (BATs) prepared in this way undergo significant morphological changes including integration into the anchoring material, alignment with the axis of attachment/strain, and compaction of the matrix. Furthermore, cell survivorship (time to failure or breaking of the construct), is related to cell type, cell density, and composition/architecture of the anchoring material. Methods: The relative gross morphology, compaction kinetics and survivorship of various cell types including tenocytes (isolated from primary human and equine explants) and osteoblasts (human bone MG63 cells) were evaluated and compared. Briefly, BATs were cast as linear collagen hydrogels (Collagel® kit, Flexcell Intl. Corp.) in Tissue Train® culture plates using 200ÁL volume and linear trough loaders beneath silicone elastomer membranes in the Tissue Train® wells that were subjected to static vacuum tension (FX-5000™ tension system). BATs developed into bioartificial tendons as the matrix was compacted by cell-driven collagen fiber and cell alignment. BATs were imaged every hour for the first 24 hours and every 6 hours thereafter using the ScanFlex™ system and serial kinetic images analyzed (XyFlex™ software) to determine change in surface area over time and/or time to failure when the BAT broke in vitro. Results: Matrix compaction rates varied in accordance with tissue origin, cells derived from fibrous connective tissue demonstrating the greatest compaction but decreased survivorship as compared to cells derived from mineralized connective tissue (bone). Furthermore, differences in the architecture/composition of the material used to facilitate anchoring of the 3D linear construct were observed to have an impact on time to failure with BATs comprised of primary tenocytes anchored to a 3D open cell foam exhibiting significantly increased survivorship as compared to identical BATs adhered to linear nonwoven mesh anchor tabs despite comparable compaction kinetics. Conclusions: Taken together, these data suggest that although various cell types are capable of matrix re-organization and compaction to form linear 3D BATs, the gross biological properties of a given BAT are largely dependent upon both cell type and architecture/composition of the anchoring material.

Novel Wound Tear Model for 3D Bioartificial Constructs In Vitro
Wimmer C, Addison K, Frazier C, Qi J, Banes AJ. 14th Annual Conference of the North Carolina Tissue Engineering and Regenerative Medicine Society, Raleigh, NC, September 10, 2012.

Wound models for connective tissues in vitro generally test for kinetics of cell migration, toxicity or apoptosis in response to a drug. We have developed a 3D wound and tear model for bioartificial tendon to test the effects of compounds that are stimulatory for tenocytes in response to traumatic injury as well as mechanical stimulation. A 6 place, trough loader was created in Solidworks with dual arm, y-shaped cavities and submitted for construction of a rapid prototyped polymeric device to fit beneath a standard BioFlex, rubber bottomed culture plate. A nonwoven nylon mesh was bonded to the membrane at the outside segments of arms and at the base of the Y with tabular extensions to capture a cell-populated hydrogel. The bottoms of the cavities have holes through which vacuum can draw down the rubber membrane to create a void for deposition of a cell-seeded collagen gel (bioartificial tendons, BATs). Once gels have compacted for three days, a modified arctangular loading post was placed beneath the membrane to apply tension across the Y arm. Strains of up to 18% can be applied to create a tear at the stress riser at the crotch of the Y with a vacuum-based system. Cells can be mechanically conditioned at low strain and low frequency or subjected to traumatic injury by high strain cycling of the construct. Live/dead cell counts DNA be made in situ or by releasing cells from the matrix and performing direct cells counts.

Human and Equine Tenocytes Localize Tenomodulin to Chromatin during Mitosis
Norman W, Kim J, Qi J, Banes AN, Banes AJ. 14th Annual Conference of the North Carolina Tissue Engineering and Regenerative Medicine Society, Raleigh, NC, September 10, 2012.

Tenomodulin is an accepted tenocyte biomarker that is co-expressed with scleraxis and Mohawk during tendon development. Tnmd is also expressed differentially in human flexor carpi radialis and biceps tendons from surgery. It is thought to be involved in anti-angiogenesis but results in a KO mouse model found only a reduction in cell proliferation in tendons. We hypothesized that Tnmd might have a role in regulation of cell division and found Tnmd localization in mitotic tenocytes. Flexor carpi radialis (FCR) or biceps tendon (BT) cells were isolated from surgical specimens. Equine superficial digital flexor tendon cells were isolated form tendons at necropsy. Cells were tested for Tnmd expression and were found to express Tnmd differentially. Cells were plated on cover slips and paused in prometaphase with 50 nM nocodazole for 16h, then released in serum-containing medium to restart the cells. Cells were assessed in prometaphase, metaphase, anaphase and telophase for Tnmd localization and DNA by DAPI stain. Tenocytes in mitosis showed robust stain for Tnmd around the chromatin and distinct co-localization with chromosomes in metaphase, anaphase and telophase. Human tenocytes from control (FCR) and pathologic (BT) tenocytes likely express different ratios of Tnmd isoforms due to the different metabolic states and rates of cell proliferation. Tnmd clearly has a role in mitotic cells as it co-localizes with chromatin and with lamin B in a peri-chromosomal locale. Given that equine tenocytes do not make I2 but do show chromatin binding, Tnmd I1 and I3 are likely binding chromatin and regulate cell proliferation.

Nuclear Localization and Chromatin Association of Tenomodulin in Human and Equine Tenocytes
Qi J, Banes AN, Dmochowski JM, Norman W, Kim J, Bynum D, Patterson M, Creighton A, Banes AJ. 14th Annual Conference of the North Carolina Tissue Engineering and Regenerative Medicine Society, Raleigh, NC, September 10, 2012.

Tenomodulin (Tnmd) is classified as a type II transmembrane glycoprotein with a BRICHOS domain, and is highly expressed in developing as well as in mature tendons. Tnmd has been reported to have anti-angiogenic properties but results in a knockout mouse model did not substantiate that claim. We have reported that Tnmd translocated into the nucleus in response to stretch and knockdown of Tnmd with RNAi, reduced cell proliferation. However, the function of Tnmd in the nucleus is still unknown. We also reported that stretch induced the expression of interleukin-1beta (IL-1beta) in tenocytes. Therefore, we hypothesized that IL-1beta might induce nuclear localization of Tnmd and that Tnmd might associate with chromatin during mitosis. Since IL-1beta is key player in tedinopathy, this study may shine a light on the treatment of tendinopathy in the future. In the present study, Tnmd showed nuclear localization at less than 8 h in response to cyclic stretching. Treatment of tenocytes with IL-1beta also induced Tnmd nuclear localization within 8 h. Tnmd showed nuclear localization in almost all of the cells at 8 h post-addition of IL-1beta. Surprisingly, immunochemically detectable Tnmd co-localized with chromatin during mitosis from prometaphase to telophase in human and equine tenocytes at 8 h post-addition of IL-1beta. Given a role in cell proliferation and a nuclear localization, we speculate that Tnmd plays a role in cell cycle regulation. Since IL-1beta is a key player in tendinopathy, this study may lead to a better treatment for tendinopathy.

Tenomodulin Associates with Chromatin at Mitosis in Tenocytes and HeLa Cells
Banes AJ, Qi J, Dmochowski JM, Banes AN, Norman W, Kim J, Bynum D, Patterson M, Creighton A. 14th Annual Conference of the North Carolina Tissue Engineering and Regenerative Medicine Society, Raleigh, NC, September 10, 2012.

Tenomodulin (Tnmd) is a tenocyte biomarker with three isoforms. Results from a knockout mouse model of isoforms I and II, as well as RNAi experiments that knocked down all three isoforms, reduced cell proliferation. Strain in vitro localized Tnmd immunochemically to the nucleus. Given a role in cell proliferation and a nuclear involvement, we hypothesized that Tnmd might have a role in cell cycle regulation. Prometaphase cells from tenocytes or HeLa cells showed robust Tnmd staining throughout the cytoplasm. Cells released at 16h from the nocodazole block showed Tnmd localization in a peri-chromatin boundary in prometaphase. One h post-block, cells in metaphase showed robust Tnmd staining in a punctate border at the periphery of the chromosomes and in the cytoplasm. At two h post-block, at anaphase, Tnmd localization was peri-chromosomal at the segregating chromosomes and in dual puncta at poles in the cell where centrioles locate. At 2.5h post-block, at telophase, Tnmd signal was less robust, still in a peri-chromosomal localization with polar puncta. GFP isoforms one and two were cytoplasmic, showing globules of GFP-Tnmd, with a focus around the nuclear envelope. GFP-isoform I had a clear overlap with anti-Tnmd antibody signal as did isoform II. GFP-isoform I was cytoplasmic at prometaphase and in a distinct halo around the chromatin, then tracked with the segregating chromosomes throughout mitosis. This observation may place Tnmd isoform I at the checkpoint complex in anaphase as a novel but dispensable regulatory protein in the cell cycle, since RNAi results demonstrated reduced cell proliferation.

The tendon biomarker tenomodulin is expressed at tenocyte levels in cancer cells derived from a uterine carcinoma (HeLa)
Kim J, Norman W, Qi J, Banes AN, Banes AJ. 14th Annual Conference of the North Carolina Tissue Engineering and Regenerative Medicine Society, Raleigh, NC, September 10, 2012.

Tenomodulin is a tenocyte biomarker used in tissue engineering, present in embryonic and adult cells. Although it is an accepted marker for tendon cells, its function is unknown. Recently it has been shown that Tnmd exists in three isoforms, none of which are secreted. Results of a KO mouse model found a reduction in cell number in tendons. We hypothesized that Tnmd might be involved in cell cycle regulation, given cell numbers in tendon are reduced in the KO mouse and tenocytes treated with RNAi to Tnmd also reduced cell proliferation. HeLa cells were tested for Tnmd expression and were found to express Tnmd as highly as tenocytes. Cells were cultured in growth medium, split and plated on cover slips and paused in pro metaphase with 300 nM nocodazole for 16h, then released in serum-containing medium to restart the cells. Cells were collected in prometaphase, metaphase, anaphase and telophase and assessed for Tnmd localization and co-localization with a-tubulin for spindle fibers, ?-tubulin for centrioles and GFP-transfected Tnmd isoforms. Immunolocalization of Tnmd showed that antibody staining was peri-chromatin in prometaphase and metaphase, then at the tip of the chromosomes and around the chromosomes in anaphase and telophase. The biomarker, tenomodulin, is not specific to tendon and is found in cancer cells, such as the continuous cell line HeLa. Its functions may involve regulating the availability of chromatin via methylation and degradation. Tnmd also has an early role in tendon, likely as a cell cycle control protein regulating tenocyte fate.

Cell Migration with and without Strain
Addison K, Frazier C, Wimmer C, Banes AJ. 14th Annual Conference of the North Carolina Tissue Engineering and Regenerative Medicine Society, Raleigh, NC, September 10, 2012.

Cell migration is a complex process that is essential in wound-healing. The methods of cellular migration include weak-adhesion locomotion or "amoeboid motion", blebbing, or organized cytoskeleton-driven motion. Currently cell migration and wound-healing assays are preformed using hard-plastic or glass lab equipment, static surfaces that do not experience strain. One important factor that affects cell migration is external strain applied to the cell and cellular matrix; this induces an increase and activation of matrix metalloproeinases (MMPs). Increases in MMP-1, 2, 3, 9, and membrane type-1 MMP count have been shown to increase cell migration rates. It is important to test and observe cell migration when exposed to varying levels and patterns of strain similar to what happens with in vivo conditions. Using Flexcell® BioFlex® culture plates, cells can be seeded using the Cell Migration trough loader. This trough loader pulls the membrane into two distinct seeding regions (4mm x 9mm) with a 1000 micron migration gap between the two regions. Migration can be observed in a control group (0% Strain), alongside a group of cells that will be exposed to strain. Using the FX5K™ system, sinusoidal and static strain regimens can be a forced on the flexible silicone membrane upon which the cells are seeded. Results from a non-strained Hela cell control group were obtained by calculating the cell covered area percentage compared to the total image area. Migration rates averaged 370 microns per day for 7 days.

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