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The force is strong with this one.
Darth Vader
The force is strong with this one.
Darth Vader
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We at Beachbody® are at war against the most formidable opponent the United States has ever faced: obesity. It's killing more people a month than our two other more high-profile combatants—terror and drugs—have killed this decade. Obesity is the Darth Vader of all the dangers we'll face in our lifetimes. Smoking once ruled our universe, but a new master has taken over the dark side. Here, in the final installment of our latest trilogy on obesity, we offer a new hope of getting the force back on our side.
As usual, we begin in what appear to be ever darkening times. Just last week, a report was released showing that an obese 10-year-old kid may have the arteries of a 45-year-old. Each time we offer a solution, the obesity empire rebuffs our efforts. Earlier this year, we released
Shaun T's Fit Kids™ Club. Vader laughed as our infant mortality rate skyrocketed compared to the rest of the world. We offered
Kathy Smith's Project:YOU! Type 2™ and then saw the number of people who either are pre-diabetic or are already diagnosed with type 2 diabetes skyrocket to nearly a third of our population. Earlier this month, we offered 10 solutions to the epidemic (see "10 Ways to Fight Obesity" in the Related Articles section below). Last week, we're told that over half our kids are projected to be obese within a generation. Admittedly, things look bleak for the rebellion. But read on and you'll see that we don't need a Jedi to guide us in this war. The force is very powerful. And it's controlled by us.
In the film Idiocracy, we're offered a glimpse at a possible future should we continue to follow our current patterns of regression. In the film, society has devolved into an anti-intellectual state completely controlled by corporate advertising interests. We're at a crossroads—we're watering our plants with a version of Gatorade, the only beverage we consume, and our plants will no longer grow. The corporate-controlled Food and Drug Administration's food pyramid features nothing but fast food, sports drinks, and mind-altering legal substances: alcohol, caffeine, and tobacco. And while it's an over-the-top comedy, one can't wonder whether the most unbelievable thing in the movie is that it's set 500 years in the future. At the rate we're going, we could be living—or at least eating—this way before the end of this millennium.
Case in point: The latest studies on obese children. At the American Heart Association's conference last week, two studies, done independently in different countries, showed that obese children had a "vascular age about 30 years older than their actual age." This, of course, greatly increases their chances for heart disease. So much so, in fact, that the American Academy for Pediatrics is now recommending cholesterol-lowering drugs for some children. "As the old saying goes, you're as old as your arteries are," said Dr. Geetha Raghuveer of Children's Hospital in Kansas City, to the Associated Press. "This is a wake-up call."
But the wake-up call needs to ring louder. How we've become so unconcerned with our health isn't a mystery. We've simply followed marketing trends, which have seen junky foods and other easily consumable items become more and more a mainstay in our diets. Gas stations and convenience stores are filled with virtually nothing but junk food and drug-filled beverages. Supermarkets are only slightly better, placing their healthy items on the fringes and filling the middle aisles with premade medleys of genetically modified corn and soy. The companies that have become successful marketing this said junk now dominate our airwaves to the point where a person dropped into an American city could easily assume that we, indeed, do live primarily on fast food, sports drinks, and mind-altering legal substances.
Our next wartime strategy is going to be to consolidate our resources and simplify. If society's attention spans are dwindling, we'll offer a simple three-point plan to unify our rebellion. With or without the help of Obi Wan, this might be our only hope.
Related Articles
"10 Ways to Fight Obesity"
"Are You at Risk for Type 2 Diabetes?"
"7 Tips for Healthy Lunches Kids Will Love"
Got something to say? Chat with the writers and other readers this Monday, November 24th, at 7:00 PM ET, 4:00 PM PT in the Beachbody Chatroom!
If you'd like to ask a question or comment on this newsletter article, click here to add a comment in the newsletter review section or you can email us at email@example.com.
Check out our Fitness Advisor's responses to your comments in Steve Edwards' Mailbag on the Message Boards. If you'd like to receive Steve Edwards' Mailbag by email, click here to subscribe to Steve's Health and Fitness Newsletter. And if you'd like to know more about Steve's views on fitness, nutrition, and outdoor sports, read his blog, The Straight Dope.
Can you turn fat into muscle? Is the morning the best time to exercise? Will you get cramps if you drink too much water when you exercise? Can those ab machines on TV get rid of your potbelly? Spend time surfing the Internet for fitness information and, pretty soon, your head will explode from reading all the contradictions and misinformation about the best strategies to get fit. Here are some of the more popular myths and old wives' tales that have been propagated over the years.
Related Articles
"Getting to the Core of Your Ab Routine"
"The Sweatiest Thing"
"What Does It Mean to Be 'Fat'?"
Got something to say? Chat with the writers and other readers this Monday, November 24th, at 7:00 PM ET, 4:00 PM PT in the Beachbody Chatroom!
True or False?
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"If you don't exercise every day you might as well not exercise at all is not just some excuse from people who dont want to exercise. I was told that by a personal trainer. Not only that he told me it was useless to exercise unless I did at least an hour a day at least five days a week. There have been a ton of magazine articles that say similar. If the people are getting bad information don't blame the people for being confused."
– Marin, Denver, CO
"if your doing a weight lifting workout isnt it better to do it in the morning because when you lift weights your muscles are still working when your done so it would go throughout the day but if you do it at night, it stops when you go to sleep"
– Mark, Surrey, BC
"Looks like someone got sold hook line and sinker by their personal trainer that was trying to make them pay for more frequent, longer workout sessions. Only thing that means is they make more money off of you."
– Henry, San Francisco, CA
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VINS celebrates 40 years of conservationBy Christian Avard
Staff Writer | December 02,2012Vyto Starinskas / Staff Photo
A red tail hawk flies out of employee Katie Christmans hands during a raptor environmental educational demonstration at the Vermont Institute of Natural Sciences on Friday.QUECHEE An Upper Valley landmark known for conservation efforts marked a major milestone this fall.
The Vermont Institute for Natural Sciences in Quechee celebrated 40 years of successful wildlife preservation and environmental education programs. VINS is a renowned environmental organization and its work has impacted the local community, the state and beyond. VINS success can be traced back to its origins when local residents took action to protect local habitat from industrial pollution.
The Ottauquechee River is a critical water source for the Upper Valley. It starts high up in the Green Mountains and meanders east until it joins the Connecticut River.
The Ottauquechee was heavily polluted in the early 1970s, when mills were dumping waste upstream. A group of Woodstock citizens got together, cleaned up the river and stopped the mills from causing further harm.
So that was the start, VINS President John Dolan said. They became successful pretty quickly. So then they thought OK, what do we do now?
VINS began as an organization in 1972 and operated out of a Woodstock storefront. They moved into a old farm building on Church Hill Road in Woodstock in the late 1970s and began new programs.
VINS conducted ornithological research and the rehabilitation of avian wildlife. The work was prompted by VINS co-founders Sally and David Laughlin, Richard Farrar, June McKnight and Jenepher Lingelbach, Dolan said.
The staff initially took in injured raptors because nobody else knew where to bring them, Dolan said. (Sally) took care of injured birds in her backyard and had a number of enclosures with injured owls, hawks and falcons that were brought to her for treatment. It really evolved into a professional rehabilitation operation.
As VINS settled in, a new program was established, Environmental Learning for the Future, an award-winning environmental education program for elementary-age children.
ELF is designed to give children an understanding of and appreciation for the natural world and increase scientific and environmental literacy. The ELF curriculum includes a textbook called Hands On Nature and is used throughout New England schools.
As VINS grew in the 1990s, so did its programs, and a new facility was needed. VINS moved to a new location near the Quechee Gorge in 2004 and built a new nature center that includes a rehabilitation and release center, exhibits, nature trails and more.
VINS established an in-house ornithological research group that studied native birds of Vermont. According to Dolan, the group was instrumental in removing the Bicknells Thrush, a songbird, and the Vermont Loon from the endangered species list.
The nature center was a significant development for VINS. It became a destination, it drew a lot of people from across the country and raised our profile, Dolan said.
The final and perhaps the most influential contribution to VINS success is the support from volunteers, donors, researchers and conservationists who shared a concern for wildlife and local habitat.
A lot of what you see at the center comes from tens of thousands of volunteers that helped us out over the last 40 years, Dolan said. Ive been with VINS for six years but I can see from where I sit the very rich legacy that staff, donors and volunteers left behind: a tremendous knowledge of natural sciences and reputation for quality programing in environmental education. VINS has always maintained high standards of quality in everything its done.
To learn more, visit www.vinsweb.org.
@rutlandherald.comMORE IN Vermont NewsMONTPELIER Officials from the Vermont Agency of Natural Resources are going to be holding a... Full StoryMONTPELIER The state Public Service Board on Friday announced it would fine Vermont Gas Systems... Full StoryMONTPELIER Less than a month after Vermonts out-of-state inmates were moved from a private... Full Story
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- RICHARD'S POOR ALMANACK: On this day 1739, 'Richard Palmer' identified in prison at York Castle as the notorious outlaw DICK TURPIN; IN 1836, Battle of the Alamo begins near San Antonio de Bexar, Texas; 1896, the Tootsie Roll invented by LEO HIRSCHFELD.
- RICHARD'S POOR ALMANACK: On this day in 1472, Orkney, Shetland islands put up as collateral by Norway to Scotland in lieu of dowry for MARGARET OF DENMARK on her marriage with JAMES III, king of Scotland; 1962, JOHN GLENN first American to orbit Earth.
- TOMORROW'S HEADLINES TODAY: City mayoral candidates debate campaign issues; Hartford, Conn., woman still missing; Neal Goswami reports attempts to legislate suicide; local woman loses 100 pounds through TOPS program.
- RICHARD'S POOR ALMANACK: On this day in 1878, JOHN TUNSTALL murdered near Lincoln, New Mexico, by the outlaw JESSE EVANS; in 1930, ELM FARM OLLIE first cow to fly in aircraft, first to be milked airborne; 1955, nuke test WASP; '79, snow in Sahara.
- TOMORROW'S HEADLINES TODAY: Rutland Herald News Editor Alan J. Keays and staff writer Gordon Dritschilo discuss stories planned for the February 18, 2015, edition of the newspaper: Winter budgets maxed, legal marijuana, Springfield bank job, USPS slowdown
- RICHARD'S POOR ALMANACK: On this day in 1249 AD, ANDRE of LONGJUMEAU is dispatched by LOUIS IX of France to meet the KHAGAN, ruler of the Mongol Empire; in 1804, during 1st Barbary War, STEPHEN DECATUR scuttles the pirate-held USS Philadelphia in Tripoli.

subset: commoncrawl


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ERZ10439 In Stock
Bring a little twist to plain old silver hoops with these beautiful 1" inside out wavy cz hoop earrings! These sophisticated silver hoops feature sparkling 1 mm czs running along the inside to the outside and have a bar closure. They are 1" in diameter. These wavy hoops look great with an everyday outfit but work equally well when paired with formal wear.
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Jemared Demario Axen, 27 — disorderly conduct, deriding a police officer
Terry Rashad Taylor, 25 — driving under the influence
Arthur Wellington Fleming, 20 — breaking and entering auto
Armando Martinez Sanchez, 44 — disregarding stop sign, no South Carolina driver's license
Melinda Janelle Roche, 29 — distribute, sell, manufacture, or possession with intent to distribute controlled substance, manufacture distribution etc. of cocaine
Zackery Caine Clippinger, 27 — attempt and conspiracy general provisions of the drug
Christopher Corley, 30 — assault and battery third degree
Jennifer Lynn Oakman, 23 — trafficking crack cocaine, possession with intent to distribute, hold per NCIC teletype, possession of drug paraphernalia, simple possession of marijuana
Mark Anthony Elmore, 48 — unlawful use of telephone
Cottie Marie Ledford, 24 — malicious injury to tree, house, outside, or fixture; trespass upon real property, theft of electric current
Marisol Santiago, 27 — assault and battery third degree
Nicole Leigh Masters, 28 — two counts shoplifting
Michael Scott Smith, 29 — possession of less than one gram of meth or cocaine
Alysia Leeann Smith, 26 — financial transaction card
Ryan Jamal Walker, 19 — unlawful carrying, possession, possession of 28 grams or less, resisting arrest
Brittany Nicole Williams, 23 — petit or simple larceny
Malik Qadir Shakur, 50 — disorderly conduct, assault and battery
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The season will continue for a pair of Lady Husky tennis players. Haley Maloney and Anna Wooten earned a third place finish last weekend in the 2A Midwest Regional held in Salisbury.
In the semifinal round, Sallie Kate Meyerhoeffer and Alexandra Drye from Salisbury High School defeated Wooten and Maloney 6-1, 6-0.
The Ashe duo bounced back quickly, and knocked off a familiar foe in the consolation third place game to move on to the state tournament. Wooten and Maloney were 6-2, 6-1 winners over Ashley Griffin and Heaven Walters of Starmount.
"I am so proud of these players and feel they can go far in the state playoffs," said head coach Larry Dix. "They have worked hard and have had a great senior year."
Entering the state tournament, Wooten and Maloney hold a record of 23 wins against just one loss. The tournament will take place this Friday and Saturday in Cary.


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Monday, April 28, 2014
A Cross High School teacher is under investigation for alleged sexual misconduct with a student.
According to the Berkeley County Sheriffs Office, the BCSO is conducting an investigation after receiving notification from the Berkeley County School District that a teacher at Cross High School had been accused of sexual misconduct with a student.
The BCSO released a statement to the media Monday night.
According to the school district, the teacher has been placed on administrative leave pending the outcome of this investigation into these allegations, the release stated.
According to the release, no further information will be available until the investigation is completed.
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Join the Experience of Pearl River Resort a spectacular blend of casino thrills, family-style fun and dazzling live entertainment with down-home Southern hospitality and warmth.
Escape to the rolling hills of Choctaw, Mississippi and engage in infinite diversions, from world-class casinos, two award-winning golf courses and marvelous restaurants to timeless and comfortable accommodations, an exquisite spa and exceptional venues for meetings and special events. Despite our extraordinary amenities, it is actually our intimate, easygoing approach to service – above all else – that sets this casino and spa resort apart as a truly special place.
Offering fun in every corner, Pearl River Resort is tucked among the towering pines and oak forests of Choctaw, Mississippi and is owned and operated by the Mississippi Band of Choctaw Indians.
Come discover everything you need for the perfect getaway at the South's premier gaming and family vacation destination.


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I agree with EW (as usual) I do have another perspective besides that tho'"Often we use the old testament as an example towards spiritual truths in the new testament. so if they are physically destroyed by God, does this example teach that unrighteous will be spiritually destroyed?" I believe that when we are consumed by the fire of Holy Father, our refinement and purification removes the dross of the sinful thoughts words and deeds we have done. A total sinner then would be born anew but would also have nothing left of the old life. (Much as aborted baby's or like children way to young to understand much of anything before they die.)We, that are called the saints of God, will cast our good works at his feet and realize in totality that all true good works were the Holy Spirit working through us. How much of the sinners personality or soul will remain - I don't know. But Job said, "I know that one day I will see God, it will be me, in my flesh, and not another." so it won't be like a totally new creature in the next age in all respects but, in some ways it will.


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"Always forwards, never backwards." Erich Honecker
J, that's beautiful !!!!! When I got ready to do an RPK I wound up having to go with the Romy, because I just couldn't afford the Yugo. That pic makes me wish I had spent the extra $$$. LOL. Very nice brother.
Enforcement, NOT Amnesty!!!!!!
"If theyre going to come here illegally, apply for & receive assistance through a corrupted Government agency encouraging this lawless behavior, work under the table & send billions of dollars each year back to their families in Mexico, while bleeding local economies dry, protest in our streets waving their Mexican flags DEMANDING rights, while I have to press 1′ for English, then they need to be shipped back to where they came from!" -Chad Miller
any rpk is a cool rpk but i definitely like the m72 for sure
"Always forwards, never backwards." Erich Honecker
Very nice
Thanks for the pics
"Government is not the solution to our problem, government is the problem" Ronald Reagan


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Playful puppies make the perfect addition to any nursery or playroom. Unique mobile is sure to be the center of attention. Measures 20" x 20."
More about Flendsted:
Flensted Mobiles is a family business that designs and creates mobiles in Denmark. Their mobiles are created so that the elements are in constant motion while the entire mobile maintains a harmonic balance. Each mobile is carefully assembled, and balanced by skillful hands.
Perfect for above the crib, over the changing table or any area of the playroom, these unique mobiles bring a sense of fun and whimsy to any space. Designed to last and be appreciated long after baby outgrows the crib.


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BERZ10013 In Stock
Looking for a beautiful and fun pair of earrings that you can wear to a family get-together or a night on the town? Well here they are! These Elegant Circle and Teardrop Chandelier Earrings are a perfect accessory to any outfit! The way they flow back and forth give them an elegant graze at you move, and the bright cz's make them shine bright! They will make you look like a million bucks, and you got them for a steal!
Eve's Addiction.com wants you to be a happy customer. We want you to be completely satisfied with the sterling silver jewelry that you receive from us, as we only sell high quality silver and cubic zirconia cz jewelry. We do understand that you may not like your purchase for some reason.
You may return or exchange your jewelry within 60 days of purchase if you are not satisfied with it. The jewelry must be returned in its original condition for a refund or exchange. Engraved, personalized, photo, monogram, name and initial, chocolate and clearance items are not returnable or exchangeable.


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From the invitation to your event's final moments, each interaction is an opportunity to engage with your audience, and we're here to help you make the most of every one.
Specializing in connecting our clients with their Latin American audiences, Forums Event Design & Production offers the cultural insights to help you capture hearts and minds, create a buzz for your products and services and yield the most valuable result—a stronger bond with your brand.
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How to be in 5 countries in one day
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text: An event you shouldn't miss. The book, "The Blessing Next to the Wound", was selected by the Amnesty International group in Pasadena as their November reading selection. No wonder. It is the story of Hector Aristizábal surviving poverty, torture by the US-trained military, cocaine cartels, and civil war in Colombia, as well as facing the challenges of exile. Co-written by Diane Lefer. Hector and Diane will be at Vroman's Books in Pasadena TONIGHT, Friday Nov 12th at 7:00pm


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Facility & Support Services
Purchasing Specialist
Scott County Administrative Center
600 W. 4th St.
Davenport, Iowa 52801-1030
(563) 326-8793
Scott County is Tax Exempt.
- Federal I.D. Number is 42-6004465.
- Illinois Tax I.D. Number is E9960-5295-05.
In keeping with the Purchasing Policy, "to provide increased economy in County purchasing activities and to maximize, to the fullest practical extent, the purchasing value of public funds," the purchasing division researches price comparisons for virtually any product or service when departments look to purchase.
Scott County Request for Bids:
Open bids expiring on date listed. [ Open Bids | Closed ]
[ 2013 → 20130718 19039 hybrid vehicle ]
July 18, 2013 19039 hybrid vehicle
- No files listed at this time.
Subscribe to Bid Notifications:
Vendors on the county bid list will be notified of opportunities to submit competitive prices for required products or services. [ Add your company to our vendor notification list ]
Purpose of Purchasing:
- The Purchasing Division maintains a large bidders list of vendors who represent nearly every product/service imaginable, and who would like to be contacted to give competitive price quotes.
- Purchasing regularly researches products and obtains price information for county departments. This assists departments in making a sound purchasing decision, gives vendors an opportunity to bid, and keeps prices low through competitive bidding.
The Scott County Purchasing Division has:
- a large number of vendors on file ready to be contacted when a department is looking to purchase.
- the latest Consumer Reports magazines on file.
- a wide variety of catalogs and brochures.


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Top 10 Slice Posts of 2012
Before leaping into a year of new pizza trends, recipes, openings, and chain restaurant shenanigans, let's take a look back at the most popular posts (according to our web analytics) of 2012. Here are the top ten most visited posts from the past year. What trends do you think 2013 will bring?
- Where to Eat Pizza in NYC: The Ultimate 15 Pizzeria Itinerary
- A Taste of the Cheeseburger Crown Crust Pizza from Pizza Hut in the Middle East
- How Is Domino's New Gluten-Free Pizza Crust?
- The Pizza Lab: Awesome Pizza Without An Oven (aka Skillet Pizza)
- The Pizza Lab: Three Doughs to Know
- Chain Reaction: Buffalo Chicken Pizza from Papa John's
- The Domino's Makeover
- Video: Jim Lahey Makes No-Knead Pizza At Home
- Pizza Madness: Pizza In a Jar
- The Pizza Lab: How To Make Fried Pizza At Home (à la Forcella)


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Sellards' leadership expands St. Mary's
HUNTINGTON -- Quality health care is important for any community, including whether it's accessible to all parts of a region and there are enough trained individuals to deliver it.
As chief executive officer of St. Mary's Medical Center, Mike Sellards is attuned to those issues. And under his leadership, St. Mary's has worked to address them.
Last year, the hospital completed construction of and completed a $18 million campus in Ironton, Ohio, which had been without around-the-clock medical care for more than a decade. The new facility includes a seven-day-a-week emergency room and has imaging and lab services, as well as specialty doctors doing rotations.
Just a few years earlier, St. Mary's transformed the former Big Bear supermarket building at 29th Street and 5th Avenue into its Center for Education. It opened in 2009, providing a state-of-the-art facility to house St. Mary's School of Nursing, School of Medical Imaging and the School of Respiratory Care -- and providing the education and training for people to deliver health care for coming generations.
For those accomplishments and others as the head of St. Mary's Medical Center, Sellards has been named a 2012 Community Impact Award winner by The Herald-Dispatch as part of its Citizen Awards recognition.
Those who nominated Sellards pointed to his savvy business IQ and his ability to successfully merge the compassion and mission of the Pallottine Sisters with a 21st century health care system.
"Mike is truly a passionate and dedicated health care professional whose tireless service, both in the health care arena and in the other community services that he provides and supports, has made a real difference to the Huntington community," said Joseph M. Letnaunchyn, president of the West Virginia Hospital Association for the past six years.
Letnaunchyn said the success of St. Mary's at the local level is tied to Sellards' active participation at the state and federal level, making decisions based not just on the current state of health care but its future.
Sellards is an active member of the association's Board of Trustees and its executive committee, a position he's held for more than 10 years. He also has served as a state delegate to the American Hospital Association Regional Policy Board.
"At the local level, I have had the opportunity to see Mike in action through his implementation of the numerous changes that have occurred at St. Mary's Medical Center, both in new services that have been made available for patients, and in the facility changes and improvements that have occurred over the years to improve access to care in the Huntington area," Letnaunchyn said.
Sellards' caring demeanor and heart for St. Mary's goes back a ways, as he was born at that very hospital. He grew up in Huntington, then went to the State University of New York in Albany, where he had some family ties. He also attended Michigan State University to earn his master's in business administration.
He said he had an eye for health care, though there was no degree at the time for administration and management. But he said there was an internship/residency program with a hospital in Albany that allowed him to see its inner workings.
"Hospitals are very complex operations," Sellards said. "The business model has to be different. It's also a mission that is unique to the business world."
In Catholic health care, he said it's a ministry, which is part of the reason why St. Mary's has seen fit to invest the way it has.
The ministry at St. Mary's really points to two areas: health care and education. The former goes to the heart of the facility in Ironton, said Bill Dingus, executive director of the Lawrence County Development Corp.
"Mike Sellards is a name well respected in the Ironton and Lawrence County area leadership community for his tireless efforts over the last six years to enhance the five community health centers operated by the Ironton/ Lawrence County Community Action Agency," Dingus said. "During the last three years, he shared in leading the effort to build an $18 million advanced medical center with trauma capacity emergency room services to serve the Ironton area at a time when the economy made the decision and effort extremely challenging."
Added Dr. Joseph Touma, who serves with Sellards on the Marshall University Board of Governors: "I'm also amazed by the new St. Mary's Medical Campus in Ironton. The creation of a new ER for the Ironton area shows St. Mary's commitment to serving the medical needs of underserved areas."
Touma also wrote of the importance of the other big investment made in The Center for Education. There, St. Mary's works on conjunction with Marshall University for three training programs, including the schools of Nursing, Medical Imaging and Respiratory Care. Last year, the first class of Marshall's School of Physical Therapy started at the Center for Education, through a long-term lease with the university.
St. Mary's also continues to lead the area in employment, with more than 2,600 people, a number close to 2,700 when counting the new Ironton campus.
"Seldom does an individual have the opportunity to make such a lasting contribution to his community and have an impact felt in so many different areas: economic development, education, health care and employment," said A. Michael Perry, member emeritus on the Marshall Board of Governors, retired banker and co-founder of Heritage Farm Museum and Village.
Sellards has been CEO since 2000, after spending time as CEO at Pleasant Valley Hospital in Point Pleasant. He said he has one more challenge to lead St. Mary's through before he retires.
"One of the reasons I remained interested in health care is it's an ever-evolving business," he said. "And certainly with the Affordable Care Act, it could change more with that bill than the advent of Medicare in the 1960s. I would like to see that through to a fascinating model, then I'll turn it over to these young kids."
Sellards and his wife, Betty, have two children, one grandchild and another on the way, both by son Rob Sellards, an attorney in Huntington, and his wife, Valerie.
The Sellards' daughter, Jennifer Brotherton, lives in Charlotte with her husband, Scott.
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Volunteer with us and join thousands of people who are making every day better for blind and partially sighted people.
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The Calico Owls Baby Bedding Collection by Bananafish features fun patchwork owls that will add cheer to any nursery. Browns, pinks, blues and greens offer a delightful color palette, and embroidered touches finish off the set. Bedding Set comes with Comforter, Crib Sheet and Dust Ruffle. Bumper sold separately.
- Comforter: 35 x 45 inches; cotton
- Crib Skirt: 15-inch drop; cotton
- Fitted Sheet: Fits standard size, 28 x 52-inch crib mattress; cotton
Machine wash cold, gentle cycle.Shipping Note: This item ships via FedEx Ground within contiguous United States only. Cannot be shipped to AK/HI, PO boxes, US territories, or APO/FPO addresses.


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Results 1 to 9 of 9
Andy Roberts BJJ - Farnborough
We have moved again!, but only next door. Expanding mat space by 50% and large male and female changing rooms. Equipment shop has also expanded, selling a range of gi's, rashguards, nutrition, gracie mag, kneepads etc.
Training is from monday to saturday which is comprised of BJJ, MMA, and Muay Thai.
At ARBJJ we have often hosted world and European champions including: Roger Gracie, Mauricio Gomez, Lucio Rodrigues (Lagarto), Jude Samuel, Nicolas Gregoriades, Nick Brooks. Recently we have also been running workshops focusing on MMA, Boxing and sport psychology with a wrestling seminar currently in the works.
With our current MMA success with fighters such as Nick Chapman we have been fortunate to have been featured on Sky sports and bravo.
YouTube - Sky Sports: Cagefighter - Gym Of The Week, Andy Roberts BJJ Academy
So new stuff and summary of the new academy:
Andy is a great instructer with an enthusiastic attitude, he goes through all the moves multiple times and really makes sure we understand them. He offers practical advise and shows how to compensate for different opponents and situations. Andy does not just instruct how to do his game but focuses on encompassing all fundamentals. Andy is flexable and any new comers are given no pressure to buy a gi and can train no gi.
Since the early days we have since added Purple and blue belt assistant instructors, so that everyone is given personal feedback and attention as well as insuring a class is never cancelled if Andy cannot be there.
Another plus is Andy's affiliation with Roger Gracie and other world champions inside and outside of the UK. The UFC also currently employ Andy as a judge for events in Europe and as far as Abu Dhabi.
Andy posts all upcoming and recent competitions on his website.
Last edited by Jadonblade; 1/08/2011 8:18am at . Reason: Moved again!
- Join Date
- Dec 2005
- Location
- Descending into absurdity
- Posts
- 6,977
Posted On:
1/03/2008 9:21pm
I think his quite committed to expanding. His a mod at http://www.efnsports.com so he may be net savvy.
New mats have been provided for the Guildford session for extra room. Andy has also added a forum to his web page for members to use in order to share information, socialise and ask questions. Andy is also using this as a way of getting feedback so he can make changes and additions where needed. Gary "the smiler" Turner (professional mma fighter [beat tank abbot]) is also going to be doing some training with us. The club is continuing to grow steadily with new faces turning up almost every week. A blue belt is regularly training with the club which helps. Andy is also setting up a training session with one of the UK's first BJJ blackbelts, in the near future.
Andy is actively seeking a permanent venue which would allow lessons 5 times a week (minimum) and a host of other options. But this would remain in the surrey area.
We have just expanded our mat space by 30% and now train tuesday, wednesday, thurs (thai), friday and saturday (thai and bjj) - Check the website for specific times. Some of the senior students are also now helping out with the beginner classes, so that everyone gets attention and gets any help they may ask for.
Registered Member
- Join Date
- May 2008
- Location
- Midlands, England
- Posts
- 208
Posted On:
4/16/2009 10:43am
Yeah was good fun!. I got raped in my first fight but was fun. The other guys did really well. The grab and pull is how I would like all comps to be.
We have moved again! but only to next door. So much much bigger. MMA team is doing very well and we are training 6 days a week. I joined the club pretty early on when we were in a tiny school storage room and we have been growing relentlessly ever since. Andy is a full time dedicated instructor, BJJ literally is his life.


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May 28, 2010
AUSTIN, Texas - Junior Reindell Cole advanced in the long jump and senior Shelese Ruffin advanced in the 400 meters to highlight day one for Cal State Northridge at the NCAA West Region Championships at the University of Texas.
Cole, who came in ranked eighth in the west region, posted a season-best of 25-11 (7.90) on his first attempt of the competition. He nearly matched the distance on his second attempt before passing on his final two attempts. Cole's fifth-place finish safely puts him through to Eugene, Ore. for the NCAA Championships.
Ruffin, who ran 53.45 to finish 13th in the 400 meters, was one of 24 to advance to the quartfinal round. Ruffin owns a career-best time of 52.82. She advances to Friday's quarterfinals which begin at 5:45 p.m. CDT.
In the first round of the women's 100 meters, sophomore Precious Watkins ran 12.14 to finish 45th but did not advance to the quarterfinals. Watkins still has a pair of relays to run on Saturday.
Sophomore Jasmine Cox also had her individual championship end in the first round of the 800 meters as she posted a time of 2:19.91 to finish 44th among 47 competitors. Cox will still run in the relays for the Matadors.
In the 10,000 meters on Thursday night, junior Lilyana Morejon finished 21st in a time of 35:22.31 which was nearly one minute slower than her season-best of 34:28.34. Only the top-12 advanced to the final of the 10,000 meters.
Despite running a career-best 1:00.37 in the 400 hurdles, LaShandra Batiste did not advance after placing 27th out of 48 hurdlers.
In the field events, freshman Tiana Webberley tied for 33rd in the pole vault after clearing 12-3.50 (3.75).
In the shot out, Whitney Hooks finished 18th with a season-best of 49-1.50 (14.97).
The Matadors struggled in the javelin as Haylee Varner finished 31st and Stephanie Ralls took 38th. Ralls, who has a career-best of 161-0, posted a best of 134-10 (41.09) while Varner fared better throwing 140-9 (42.91).
On the men's side on Thursday, Cole was the lone Matador to advance. In Northridge's lone track event of the day, the 100 meters, David Reid finished 38th in 10.81 and Big West champion Bradley Ike finished a disappointing 46th in 11.01.
In the shot put, Alex Butler finished 18th with a best of 55-8.50 (16.98) while Big West champion Derek Cooper did not compete.
In the javelin, conference runner-up Lance Gonzales placed 26th with a best throw of 196-1 (59.78).
Action at the NCAA West Region Championships continue on Friday with Butler in the men's hammer, Gonzales in the men's high jump, Michael Woodham in the 110 meter hurdles, Ruffin in the long jump, and Cole and Reid in the first round of the 200 meters.


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Help Desk
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We aim to provide the highest achievable quality of technology services and support to meet the academic and administrative needs of the Faculty and Staff at Bemidji State University.
Location: Deputy Hall 161
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All use of University computers and information technology resources is subject to the MnSCU Acceptable Use Policy. Faculty and Staff are responsible for being aware of this policy and complying with it.


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Emily Blackman, Ph. D., is in her sixth season at the University of Tennessee at Chattanooga and serves as the Associate Athletics Director for Student Support Services. Dr. Blackman currently oversees all of the academic support for the Mocs student-athletes and is the sport administrator for the Men's and Women's Tennis programs. She began her current position at UTC in January 2010.
The Assistant Athletics Director for Student Support Services was a new position created to oversee and enhance the academic guidance given to Mocs student-athletes. Dr. Blackman oversees the Coordinator of Academic Services as well as the academic advisors in the athletics department. She was promoted to Associate Athletics Director in September 2012.
The Mocs have set or tied school-records for student-athlete grade-point-averages in seven of Dr. Blackman's 11 semesters at UTC. This includes a school-record 3.07 overall g.p.a. in the spring of 2014. Over half of all UTC student-athletes have made the Athletics Director's Honor Roll with a 3.0 g.p.a. greater in each term since she joined the Mocs.
Additionally, all 17 of Chattanooga's sports received some form of team or individual academic recognition during the 2013-14 school year. The Mocs also set a school record with 11 teams posting a 3.0 g.p.a. in each of the last four semesters.
Dr. Blackman first came to Chattanooga in June 2009, serving as an advisor in the Center for Advisement and Student Success. She was part of a new and innovative team of advisors that helped to integrate the largest freshman class in school history into university life.
Prior to that, Dr. Blackman worked in the Registrar's Office at the University of New Mexico as the Athletic Specialist. She was responsible for athletic admissions, certifying NCAA eligibility, progress toward degree and APR. She also worked in the UNM Athletic Compliance Office for three years and was involved with the CHAMPS Life Skills program and football advisement during her tenure.
Dr. Blackman was employed with the Detroit Lions for five seasons, working with in both the player personnel and salary cap departments. She also gained valuable experience working at the Joe Louis Arena with indoor soccer, the Detroit Red Wings and at Tiger Stadium.
Originally from Somers, Conn, Dr. Blackman earned her bachelor's degree in Health Promotion from Auburn University in 1997. She completed her master's degree in Sport Administration from Wayne State University in 2000 and her M.B.A. from UNM 2005. She completed her Ph.D. in Sport Administration from New Mexico in 2008.
Emily and her husband, Jay, were married in May of 2006. He is the Mocs Associate A.D. for Communications and Marketing. They have three daughters, Talley LaRee, Elin Elizabeth, and Nora Aycock.
Update June 2014


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Woman accused of misusing bank cards, checksAugust 06,2014BENNINGTON A Cambridge, N.Y., woman was charged with multiple felonies Tuesday after police said they believed she had misused other peoples debit cards and one mans checking account.
Lynn Senecal, 43, pleaded innocent Tuesday in Bennington criminal court to 11 felony counts of forgery and seven misdemeanor charges of using false tokens in one case. She also pleaded innocent Tuesday in the same court to a misdemeanor charge of using false tokens in a second case.
Officer David Rowland of the Bennington Police Department said in the first case that he met with a man Jan. 15 who said he used to have a relationship with Senecal. The man said that the relationship was over but Senecal was still using his credit card and writing checks against his bank account.
Rowland said there was more than $700 charged to the credit card, mostly in December, and about $2,500 in checks cashed in April and May 2013.
In the second case, Trooper Christopher Burnett of the Vermont State Police said he met with another man who said Senecal was using his bank card without permission. Burnett did not give a total but said one charge, for $89.25, was for a body piercing.
Rowland said he learned Senecal was in jail in Washington County, N.Y., in March. Rowland said Senecal told him she was being held in connection with a case involving the use of a stolen credit card but said she did not provide any more specifics.
On Tuesday, Senecal was released without bail.MORE IN This Just InNEW YORK Yes, your mutual fund may own some Greek stocks. No, most likely not a lot. Full StoryWHITE RIVER JUNCTION A Springfield man who was captured Friday evening after he took off... Full StoryLoan program launched for owners of flood damaged apartment houses Full Story
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NMSU Aggies are Tough Enough to Wear Pink is a volunteer group dedicated to raising breast cancer awareness and raising funds on behalf of Cowboys for Cancer Research, a 501(c)3 not-for-profit corporation in Las Cruces, NM.
With institutional support from New Mexico State University, our annual "pink" events have become part of the culture here in Las Cruces. And with the financial support of Memorial Medical, MountainView Regional Medical Center, the Las Cruces Sun News and 100s of local businesses and organizations, more than $3 million in cash and in-kind contributions have been presented to C4CR since 2007.
100% of the cash funds we raise remain here in New Mexico. After expenses, cash gifts are made available to cancer researchers at New Mexico State University and at the UNM Cancer Center. The balance of the annual cash generated is invested in endowment funds at the New Mexico State University Foundation and UNM Cancer Center.
We're proud to help support the collaborative research work of Drs. Jeffrey B. Arterburn of New Mexico State University and Eric R. Prossnitz of the University of New Mexico. Breakthrough discoveries in the work of Drs. Arterburn and Prossnitz and their teams have been internationally recognized in the fight against human cancers and other diseases.
We're also proud to recognized as the #1 "Tough Enough to Wear Pink" event in the world!


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A composite living skin equivalent is described comprising an epidermal layer of cultured keratinocyte cells, a layer of non-porous collagen, and a dermal layer of cultured fibroblast cells in a porous cross-linked collagen sponge matrix. Preferably the non-porous collagen is Type 1, Type 3 or mixtures of Types 1 and 3 bovine collagen, which has been pepsin treated. A process for preparing the skin equivalent is described, as well as a test kit for in vitro testing of the skin equivalent. The skin equivalent has use for skin grafting as well as in vitro testing of the effects of various substances on skin.
The questions raised in reexamination request No. 90/003,418, filed Apr. 25, 1994, have been considered and the results thereof are reflected in this reissue patent which constitutes the reexamination certificate required by 35 U.S.C. 307 as provided in 37 CFR 1.570(e).
1. A composite living skin equivalent which comprises relative to a horizontal plane:
a) a sponge first layer comprising a cross-linked collagen sponge, said first layer having upper and lower surfaces, said sponge containing cultured fibroblast cells therein,
b) a .Iadd.non-porous .Iaddend.second layer comprising a high purity .[.non-porous.]. collagen .Iadd.essentially free of exogenous glycosaminoglycans.Iaddend., said second layer having upper and lower surfaces, the lower surface thereof being in contact with the upper surface of said first layer, and
c) a layer comprising cultured keratinocyte cells in contact with the upper surface of said non-porous collagen second layer.
2. The composite living skin equivalent of claim 1 wherein the collagen of the non-porous second layer is polymerized.
3. The composite living skin equivlent of claim .[.2.]. .Iadd.14 .Iaddend.wherein the collagen of at least one of the layers is bovine source derived.
4. The composite living skin equivalent of claim 3, wherein the non-porous collagen is selected from the group consisting of Type 1 collagen, Type 3 collagen, and mixtures thereof.
5. The composite living skin equivalent of claim 4, wherein the non-porous collagen has been highly purified by pre-treatment with pepsin.
6. The composite living skin equivalent of claim 5 wherein the pores of the cross-linked first layer of collagen are of sufficient size to permit the growth of fibroblast cells therein.
7. The composite living skin equivalent of claim 6 wherein the non-porous collagen second layer is applied to said upper surface of said second layer as a thin film from a solution of collagen.
8. The composite living skin equivalent of claim 7 wherein the second layer of collagen contacts the first layer of collagen only on the upper surface of said first layer.
9. The composite living skin equivalent of claim 8 wherein the fibroblast and keratinocytes cells are human source derived.
10. The composite living skin equivalent of claim 9, wherein the fibroblast cells are obtained from treating a dermal sample with collagenase.
11. The composite living skin equivalent of claim 10, wherein the layer of keratinocyte cells is prepared by obtaining a single-cell suspension of keratinocytes, and evenly and discontinuously distributing drops of the suspension onto the layer of non-porous collagen, which is then incubated. .Iadd.
12. A composite living skin equivalent which comprises relative to a horizontal plane:
a) a sponge first layer comprising a cross-linked collagen sponge, said first layer having upper and lower surfaces, said sponge containing cultured fibroblast cells therein,
b) a non-porous gel second layer comprising a high purity collagen essentially free of exogenous glycosaminoglycans, said second layer having upper and lower surfaces, the lower surface thereof being in contact with the upper surface of said first layer, and
c) a layer comprising cultured keratinocyte cells in contact with the upper surface of said non-porous collagen second layer. .Iadd.13. The composite living skin equivalent of claim 12 wherein said sponge first layer comprising a cross-linked collagen sponge is essentially free of exogenous
glycosaminoglycans. .Iaddend..Iadd.14. The composite living skin equivalent of claim 13 wherein said sponge first layer is derived from collagen which is essentially free of exogenous denatured collagen. .Iaddend.
The present invention relates to living skin equivalents and, in particular, to composite living skin equivalents comprising an epidermal layer of cultured keratinocyte cells, a layer of highly purified, non-porous collagen and a dermal layer of cultured fibroblast cells in a porous, cross-linked collagen sponge. The invention also relates to a method of preparing the composite living skin equivalent.
Skin equivalents have many uses not only as a replacement for human or animal skin for skin grafting, but also as test skin for determining the effects of pharmaceutical substances and cosmetics on skin.
A major difficulty in pharmacological, chemical and cosmetic testing is the difficulties in determining the efficacy and safety of the products on skin. One advantage of the skin equivalents of the invention is their use as an indicator of the effects produced by such substances through in vitro testing on test skin.
Also, skin grafting of denuded areas, of granulating wounds and of burns, still present major healing problems despite advances in grafting techniques. Split thickness autografts and epidermal autografts (cultured autogenic keratinocytes) have been used with variable success. However, both forms of treatment have many disadvantages. For example, split-thickness autografts are generally unavailable in large body surface area (BSA) burns, cause further injury to the patient, are of limited use in the treatment of patients with Dystrophic Epidermolysis bullosa (DEB), show limited tissue expansion, require repeated surgical operations and protracted hospitalization and give rise to undesirable cosmetic results. Epidermal autografts require time to be produced, have a low success ("take") rate of between 30-50%, often form spontaneous blisters, are fragile and difficult to handle, exhibit contraction to 60-70% of their original size, are vulnerable during approximately the first 15 days after grafting and are of virtually no use in the treatment of deep burns where both the dermis and epidermis have been destroyed.
An alternative form of treatment is epidermal allografts (cultured allogenic keratinocytes). American researchers have treated patients with second degree burns by grafting epidermal allografts onto wounds with some success. The benefits of such an allograft include a ready supply of such grafts can be maintained so that the patients might be covered in a single procedure with a material which allows permanent healing to occur, it eliminates autografting which increases the area of wounds and leaves painful infection-prone donor sites, burn wounds covered with cultured allografts heal as quickly as burn wounds that have been covered with autografts, and enables the treatment of patients with DEB.
However, epidermal allografts still experience many of the limitations of epidermal autografts.
Full thickness skin injuries from burns destroy both the epidermis and dermis, and treatment with cultured skin needs to replace both of these components.
Hansborough, J. F and Boyce, S. T (JAMA 1989, 2125-2130) reported the application of auto epidermal cells onto a dermal equivalent which is then grafted onto a wound. The main disadvantage of this method lies in the preparation of the dermal equivalent.
Furthermore, this method involved the use of chondroitin-6-sulfate (GAG) which has weak bonding to the collagen at neutral pH, and thus may be released into the wound environment causing unforeseen long term effects on human subjects. GAG has been reported to increase scar formation in wounds which is something to be avoided in grafts. Another effect of GAG containing collagen sponges consisting in reduction of collagen blood clotting capacity can be considered rather unfavourable for application in bleeding wounds. Fibrin clot contributes to an adhesion of the graft to the wound.
Also, in this method, the collagen sponge is stabilized by being crosslinked with with 0.25% glutaraldehyde (GTA). Such crosslinked collagen is resorbed at a slower rate and is resistant to bacterial or fungal infection. At the same time the ingrowth of cells, infiltrating the GTA crosslinked collagen matrix is less. Collagen crosslinked with GTA may retain this agent as a high molecular weight polymer which is continuously hydrolyzed and monomeric GTA is released and detactable for up to 6 weeks. The cytotoxic effect of GTA on fibroblasts in tissue culture suggests that it is not an ideal crosslinking agent for a dermal equivalent which is infiltrated by hosts cells and in which the bovine collagen matrix is rapidly degraded thus releasing GTA into body fluids.
Recently, living skin equivalent grafts comprising a dermal layer of rat fibroblast cells cast in soluble collagen and a epidermal layer of cultured rat keratinocytes were successfully grafted as allografts onto Sprague Dawley rats by Bell et.al. (Journal Investigative Dermatology, 1983; 81:2s-10s). Histological examination of the graft revealed that the epidermal layer had fully differentiated to form desmosomes, tonofilaments, keratohyalin and a basement lamella. However, attempts to reproduce the living skin equivalent using human fibroblasts and keratinocytes met only with limited success. The keratinocytes failed to fully differentiate to form a basement lamella and the dermo-epidermal function was a straight line.
Problems of epidermalizing the surface of the above dermal equivalent resulted in Bell modifying his method by using skin biopsies as a source of keratinocytes, as described in the International PCT application, published as WO 86/02273. The problems with this approach are that the skin substitute thus produced is not uniform over its entire surface, since the biopsy, including the dermal portion, remains inserted in the dermis substitute. Also, the surface-area of the skin substitute obtained is limited by the number of biopsies that may be taken from a single person. The biopsy taking involve a medical procedure with potential problem of infection and scar formation. The punch biopsies of skin equivalent as a means of expanding the production of additional skin equivalent is a time consuming process. As a result, such a skin equivalent has not been used clinically. In 1988, a paper in the journal "Burns" by Colounb et al reported a 100% failure rate using Bell's method.
Bernard et al, the inventors of Australian Patent Application AU-A-13743/88 try to avoid the above problems by utilizing the culturing capacity of keratinocytes contained in the sheath of a hair follicle. The skin biopsy is replaced by a hair follicle enclosed in its sheath, which is implanted in a perpendicular position in the free surface of dermis substitute being formed. The main criticism of the above invention is the nature of the dermis and the fact that the period of epidermal growth may take several months.
Thus, there is a need for the development of living skin equivalent grafts comprising both the epidermal and dermal layers that can be easily prepared and maintained in sufficient quantities to enable a single treatment of skin wounds.
In developing a living skin equivalent it is desirable that it comprise at least some or all of the following features: it should enable rapid and sustained adherance to the wound surface, it should be tissue compatable, it should have an inner surface in contact with the wound surface that promotes the ingrowth of fibrovascular tissue, and/or it should provide protection from infection and prevention of fluid loss.
It is therefore an object of the present invention to provide cultured living skin equivalents that exhibit at least some of these features and which will substantially overcome, or ameliorate one or more of the abovementioned problems.
One aspect of the present invention concerns composite living skin equivalents comprising an epidermal layer of cultured keratinocyte cells, a layer of high purity, non-porous collagen and a dermal layer of cultured fibroblast cells in a porous, cross-linked collagen sponge.
Another aspect of the present invention involves a process for the preparation of composite living skin equivalents which comprises: obtaining a skin sample, treating the skin sample enzymically to separate the epidermis from the dermis, treating the epidermis enzymically to release the keratinocyte cells, culturing the epidermal keratinocytes until confluence, in parallel, or separately, treating the dermis enzymatically to release the fibroblast cells, culturing the fibroblasts cells until sub-confluence, inoculating a porous, cross-linked collagen sponge membrane with the cultured fibroblast cells, incubating the inoculated collagen sponge on its surface to allow the growth of the fibroblast cells throughout the collagen sponge, inverting the collagen sponge, and laminating the other surface of the sponge with a thin layer of high purity, preferably pepsin treated, nonporous collagen to form a layered sponge complex, incubating the complex to polymerize the non-porous collagen, and then inoculating it with cultured keratinocyte cells, and incubating the composite skin equivalent complex to promote the growth of the cells.
Preferably the primary culture of fibroblasts is prepared by: obtaining a dermal sample, suspending the dermal sample in a solution of collagenase to release the fibroblast cells, incubating the culture, centrifuging the culture to produce a cell pellet of fibroblasts, removing the culture medium, washing the cell pellet to remove extraneous culture medium, determining the cell number and viability, inoculating culture flasks with the cells and culturing to subconfluence.
Preferably the non-porous, highly purified collagen is selected from Type 1, type 3, or mixtures of type 1 and type 3 collagen. For example, a suitable bovine hide collagen is that obtainable from Sigma.
The collagen is purified ideally by treatment with pepsin, to remove antigenic substances.
The collagen sponge used can be any suitable such collagen sponge, for example that obtainable from Mitaplast.
The keratinocytes used in the invention are preferably prepared by the "drop" method. The keratinocytes are released from the epidermis to provide a single cell suspension. Separate drops of this suspension are spotted evenly on culture media and incubated, so that the cells spread and eventually coalesce.
The invention will now be described by way of non-limiting examples and with reference to the accompanying illustrations wherein;
FIG. 1 is a schematic representation of the process for producing the composite living skin equivalents of the present invention.
FIG. 2 a) shows the histological appearance of the composite living skin equivalent .[.2 weeks after grafting,.]. .Iadd.shown in the 1987 Annual Report of the Organogenesis Company, 150 Dan Road, Canton, Mass. 02021 .Iaddend.compared to FIG. b) which shows the histological appearance of normal skin .Iadd.as shown in that same Report. .Iaddend.
FIG. 3 shows the histology of a skin biopsy from a graft using the skin equivalent of the invention.
FIG. 4 shows the composite skin equivalent of the invention.
FIG. 5 shows a biopsy of the skin equivalent in a skin graft from a human subject.
FIG. 1 shows schematically the process for the preparation of the composite living skin equivalent.
In FIG. 1, a skin sample is separated into dermis being used as a source of fibroblasts (10), and the epidermis being used as a source of keratinocytes (11). The cultured fibroblasts (10) are inoculated into the collagen sponge (12), and after the fibroblasts colonise the sponge, the other side off the sponge is inoculated with the pepsin treated non-porous collagen. After further incubation, the non-porous collagen is inoculated with the keratinocyte culture, preferably by the "drop" method, and cultured, to eventually produce the skin equivalent (13).
Any skin sample amenable to cell culture techniques can be used in accordance with the present invention. The skin samples may be autogenic or allogenic.
Skin samples are treated with trypsin to separate the epidermis from the dermis (Eisinger, M. Method in Skin Research, Editor D. Skerrow, (1985) pp 193). The epidermis is minced and treated with trypsin to release the keratinocyte cells. The keratinocyte cells are cultured until confluence using standard methods. In a preferred aspect, the keratinocyte cells are cultured as single cell suspensions until confluence.
Primary cultures of fibroblast cells for use in accordance with the present invention may be prepared using standard methods such as, for example, the method disclosed in "A specific collagenase from Rabbit fibroblasts in monolayer culture" (Journal of Biochemistry (1974) 137, 373-385). Preferably, the primary cultures of fibroblast cells are prepared by the following method.
A dermal sample is cut up into 1 mm cubes and is suspended in a solution of collagenase buffered with Tris-HCl pH 7.4. Dermal samples may be allogenic or autogenic. A suitable collagenaise is Clostridium histolyticum collagenase. The dermal sample is preferably suspended in solution at a concentration of 1 microgram/ml. The suspension is incubated and then centrifuged at 1,500 rev/sec to remove the cells from solution. The suspension is preferably incubated for 30 minutes. The cell pellet is washed with DMEM and the number of fibroblasts cells is determined with a heamocytometer. The viability of the fibroblast cells is determined by dye exclusion using Trypan Blue. The fibroblast cells may be injected directly into collagen sponges or are used to seed culture flasks and grown to sub-confluence using standard methods. Surprisingly, this method is shorter by up to 2 weeks than other known methods which take up to 3 weeks to prepare primary cultures of fibroblasts.
The above culturing method also surprisingly yields other dermal epithelical cells which have a potential to develop into sweat glands or other skin cell types.
Cross-linked, bovine collagen sponge membranes are obtained commercially and are stored frozen. Before use, the sponges are washed with sterile water and dehydrated at 37� C. to compact the membranes.
The collagen sponge is inoculated with cultured fibroblast cells. In one embodiment, the sponge is inoculated with subconfluent cell culture. Preferably, the fibroblast inoculum has a density of about 4�105 cells/ml.
The inoculated sponge is incubated using standard methods to enable the growth of the fibroblast cells throughout the collagen matrix. In one embodiment, the sponge is incubated at 37� C., with 5% CO2 and saturated humidity for 10 days in DMEM and conditioned media. The DMEM and conditioned media is preferably changed every second day. Conditioned media is DMEM which has been used in the culture of human keratinocytes over a period of 2 days. It contains various macromolecules secreted by the keratinocytes which are known to stimulate fibroblasts. The conditioned media can be stored frozen until use. It is used in conjunction with fresh DMEM in a ratio of 1:2.
The sponge is then inverted and the upper surface is laminated with pepsin-treated, non-porous collagen. In one embodiment of the present invention, the laminate layer is (purified) pepsin-treated, non-porous, sterile type 1 bovine collagen, or a mixture of type 1 and 3 bovine collagen. The bovine collagen is obtained commercially in substantially purified form. It is treated with pepsin using the method of Drake, M. P., Davison, P. F. and Bump, S. (1966) Biochemistry 5:301-312, to remove teliopeptides. The pH of the collagen solution is adjusted to neutral pH and is sterilized with Gamma radiation. A suitable concentration of the collagen solution is 2 mg/ml. The collagen is layered as a thin film onto the sponge and is incubated to complete polymerization of the collagen at 37� C. for 60 minutes.
Cultured keratinocytes are then inoculated onto the laminate layer. Preferably, the cells are inoculated with drops of media containing cells at a density of 1�105 cells/drop. The sponge is then incubated in DMEM supplemented with 2% fetal bovine serum, .[.10 mg/ml.]. .Iadd.10 ng/ml .Iaddend.human epidermal growth factor, 0.4 mg/ml hydrocortisone and 10-9 M cholera toxin at pH 7.2 and 35� C. for 10 days.
The skin equivalent remains immersed in the above culture medium throughout the incubation period.
Prior to the clinical or in vitro application of the composite living skin equivalent, medium without bovine pituitary extract is added to the culture medium.
Surprisingly, in clinical applications, the composite living skin equivalent of the present invention results in a rapid normalization of the interface between the epidermis and dermis of the healed transplants. Histological examination of the skin equivalent of the present invention after 2 weeks culture in vitro revealed a multilayered epidermis growing on a homogeneous membrane. Histological examination of a biopsy sample of a skin equivalent of the present invention taken 2 weeks after grafting confirmed the formation of stratun corneum with a fully keratinized epidermis on a dermis populated by non-inflammatory connective tissue cells .[.(refer FIG. 2a).].. Significantly there is an undulating and morphologically mature epidermal-dermal junction when viewed by light and electron-microscopy. There was an interdigitations of rete ridges and dermal papillae that are characteristic of normal skin and are important if the interface between the epidermis and the dermis are to be normalized.
If the skin equivalent is cultured initially immersed for 4 days in the DMEM culture medium with a Ca++ of 0.05�10-3 M and then for 10 days with the epidermis exposed (air liquid interphase) in DMEM medium containing a raised concentration of Ca++ of 0.05�10-3 M then its possible to detect a multilayered, differentiated epidermis which consists of a basal cell layer on a well developed basal lamina and several layers of suprabasal cells which show early keratinization.
The above in vitro system closely resembles human skin instructural arrangement and biosynthetic output and may be adapted to test the safety of chemicals pesticides and skin irritation of cosmetic substances. Test substances applied to the surface of this system may cause irritation of the cells and trigger the release of mediators of the inflammatory response which can be measured in the culture fluid. This system may be suitable for in vitro cytotoxicity testing: the total cellular-protein assay and the neutral red uptake assay which measure protein concentration in cells provide a quantifiable assay for the presence of living and dead cells.
The skin equivalent can be cultured in glass bottles or dishes, and the various chemicals to be tested applied to this cultured skin. By continuing to supply suitable culture media the skin will continue to grow until the test is complete.
The collagen sponge provides a temporary matrix which enables the rapid and sustained adherence of the composite living skin equivalent to a wound surface, in a human or animal, which provides a surface in contact with the wound surface that allows fibrovascular ingrowth from the wound surface and which allows for neodermal structures to develop. The collagen matrix is altered and gradually broken down and replaced by endogenous collagen as the fibroblasts interact with it physically and contribute to it biosynthetically. However, as the collagen sponges are cross-linked collagen, they do not contract and so can be stored for some time, and do not exhibit contraction when grafted onto wounds.
The pepsin-treated, non-porous collagen placed on the upper surface of the collagen sponge prevents invasion of the collagen sponge by cultured keratinocyte cells and thus ensures the compartmentalization of the skin equivalent into epidermal and dermal layers. This laminate layer is also gradually broken down to allow the normal interface between the dermis and epidermis to form.
The composite living skin equivalents of the present invention are approximately 0.8 mm thick and are therefore stronger than epidermal grafts and more easily handled during grafting. The success ("take") rate for the skin equivalents of the present invention is approximately 90%, which may be attributed to the presence of the dermal layer which promotes development of vascularization of the graft. Finally, the composite living skin equivalents of the present invention enables the graft to be applied in one stage, thereby requiring a only a single graft acceptance event. To facilitate transport and application of the composite living skin equivalent, vaseline gauze may be placed over the cultured graft.
The fibroblasts and keratinocytes used in accordance with the present invention may be either autogenic or allogenic. The use of allogenic cells enables the production and storage of the living skin equivalent of the present invention thereby avoiding delays in procuring grafts for the treatment of wounds. Both cell types, keratinocytes and fibroblasts could be stores frozen for months as single cell suspensions, using published methods. After thawing these cells were viable and grew readily in culture and thus were suitable to be sued for the production of skin equivalent. The skin equivalent has been grafted successfully to a human volunteer. Since cooling supresses the immunogenicity of skin grafts (Baldwin WM et al. Transplantation 1973; 15:419-422) cryopreservation of allogenic cells helps in attenuating some undesirable antigens. The availability of composite living skin equivalents for the treatment of surface area wounds can provide a virtually unlimited quantity of graftable skin as a source of permanent wound coverage.
The present invention will now be described in the following examples using human allogenic skin cells.
Composite grafts were made from separate, parallel cultures of allogenic human keratinocytes (HK) and human fibroblasts (HF) and a cellular bovine collagen membrane. The dermal membrane was modified using type 1 bovine collagen to provide a planar surface for cultured HKs.
Human skin was obtained from surgical specimens i.e. neonatal circumcision (foreskin). After the excision the skin was placed in a sterile container with Dulbecco's modified Eagle's medium (DMEM). Specimens were delivered within a short time to the tissue culture laboratory where the epidermis was separated from the dermis enzymically according to the method of Eisinger, M (Method in Skin Research, Editor D. Skerrow 1985 p193).
Keratinocytes were cultured as a single cell suspension by the method of Eisinger which was modified as follows: HKs were cultured in DMEM supplemented with 2% fetal bovine serum, epidermal growth factor, insulin and hydrocortisone at pH 7.2. Confluent cultures were ready for harvest after 12 to 14 days either for subculture or for inoculation onto the collagen membrane.
Fibroblasts were released from dermal fragments by digesting these with Clostridium histolyticum collagenase. HFs were then grown in DMEM using standard methods.
The collagen sponge members (6 cm diameter), stored frozen in Petri dishes, were washed with sterile water and dehydrated in an incubator at 37� C. in order to compact the membranes. The sponges were then incubated overnight in DMEM supplemented with conditioned medium. Subconfluent cultures of HFs were innoculated at a density of 4�105 cells/ml onto the surface of the sponge. The sponge was then placed in DMEM in an incubator at 37� C., 5% carbon dioxide and saturated humidity for 10 days.
Medium was changed every second day. At the end of that period the sponges were turned over and a nonporous surface was prepared to provide a planar surface for cultured HKs.
Lamination was performed by applying a thin film of pepsin-treated, non-porous, sterile type 1 bovine collagen. The films were prepared from commercially available purified type 1 collagen which was pepsinized to remove teliopeptides and sterilized by Gamma radiation. The collagen solution was brought to neutral pH and applied as a thin film onto the surface of the collagen sponge and incubated for 60 minutes at 37� C.
Cultured HKs were then innoculated in drops at a density of 1�105 per drop on to the nonporous surface of the sponge and incubated for 10 days in DMEM with supplements mentioned above. Before clinical application culture medium without bovine pituitary extract was added to the culture dishes. Vaseline gauze was placed over the cultured graft to facilitate the transporting and securing of the graft to the wound bed.
In 8 operations performed on 5 children using the living skin equivalents of the present invention; the success rate was 90%.
Clinical and histological appearance of the above grafts in the eight operations of the DEB children suggested that there was no rejection.
However, in order to determine direct evidence of the long term survival of cultured allografts or their contribution to permanent epithelium, clinical trials were performed on healthy adult volunteers described in the o following example.
Composite grafts and allografts were derived the same way as outlined in Example 1. Having providing a planar surface for cultured keratinocytes, human keratinocytes attached to the surface of the cross linked bovine collagen substrate. This "skin culture sandwich" was then transplanted on bloc on to the wound bed of human patients where the collagen matrix served as a framework to allow fibrovascular ingrowth from the underlying wound bed thus permitting human keratinocytes survival, and the formation of the neodermal structures.
Clinical studies were performed on four volunteers for the removal of decorative tatoos. Each volunteer had four skin areas excised on an arm. In two areas, the skin was excised down to the fat while the remaining two excisions left dermal elements in place. Full depth and partial thickness excissions were grafted with cultured skin and split thickness autograft skin (as controls). Weekly dressings were performed until the wounds were completely healed. The wounds were photographed, biopsies were performed for histology and DNA fingerprinting.
Volunteer 1 had two areas grafted with cultured skin and the other two with an autograph. After 2 weeks the grafts had taken and there was no evidence of graft contraction. Biopsies of the central area of the grafts had a well differentiated and multilayered epidermis, and the dermis contained numerous fibroblasts and non-inflamatory cells in a loosely packed collagen matrix. FIGS. 3 and 5 show the histology of a skin biopsy from a cultured graft, 2 weeks post-operatively. FIG. 4 shows the skin equivalent before grafting. DNA fingerprinting indicated that there was a mixed population of cells both donor and recipient indicating that some of the dermal cells were derived from the patients since there was evidence of marked dermal cellularity.
Volunteer 2. The graft did not take in any of the areas. At three weeks there was histological evidence of a granulating wound.
Volunteer 3. After 4 weeks all the grafts had healed without any contraction. From a histology examination, there was a fully differentiated epidermis and a hypecellular dermis. DNA fingerprinting showed a mixed population of donor and recipient cells.
Volunteer 4. After 8 weeks the graft showed some contraction (approximately 15-20%) with the central graft area of this patient had a fully differentiated epidermis and a mature dermis without adnexae.
It should be obvious to persons skilled in the art that numerous variations and modifications could be made to the invention as described above, without departing from the overall scope and spirit of the present invention.
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