The Lost Symbol Chapter 129-131

CHAPTER 129 Robert Langdon stood mesmerized at the glass portal, absorbing the power of the landscape below him. Having ascended unknowingly hundreds of feet into the air, he was now admiring one of the most spectacular vistas he had ever seen. The shining dome of the U.S. Capitol rose like a mountain at the east end of the National Mall. On either side of the building, two parallel lines of light stretched toward him . . . the illuminated facades of the Smithsonian museums . . . beacons of art, history, science, culture. Langdon now realized to his astonishment that much of what Peter had declared to be true . . . was in fact true. There is indeed a winding staircase . . . descending hundreds of feet beneath a massive stone. The huge capstone of this obelisk sat directly over his head, and Langdon now recalled a forgotten bit of trivia that seemed to have eerie relevance: the capstone of the Washington Monument weighed precisely thirty-three hundred pounds. Again, the number 33. More startling, however, was the knowledge that this capstone's ultimate peak, the zenith of this obelisk, was crowned by a tiny, polished tip of aluminum–a metal as precious as gold in its day. The shining apex of the Washington Monument was only about a foot tall, the same size as the Masonic Pyramid. Incredibly, this small metal pyramid bore a famous engraving–Laus Deo– and Langdon suddenly understood. This is the true message of the base of the stone pyramid. The seven symbols are a transliteration! The simplest of ciphers. The symbols are letters. The stonemason's square–L The element gold–AU The Greek Sigma–S The Greek Delta–D Alchemical mercury–E The Ouroboros–O â€Å"Laus Deo,† Langdon whispered. The well-known Latin phrase–meaning â€Å"praise God†Ã¢â‚¬â€œwas inscribed on the tip of the Washington Monument in script letters only one inch tall. On full display . . . and yet invisible to all. Laus Deo. â€Å"Praise God,† Peter said behind him, flipping on the soft lighting in the chamber. â€Å"The Masonic Pyramid's final code.† Langdon turned. His friend was grinning broadly, and Langdon recalled that Peter had actually spoken the words â€Å"praise God† earlier inside the Masonic library. And I still missed it. Langdon felt a chill to realize how apt it was that the legendary Masonic Pyramid had guided him here . . . to America's great obelisk–the symbol of ancient mystical wisdom–rising toward the heavens at the heart of a nation. In a state of wonder, Langdon began moving counterclockwise around the perimeter of the tiny square room, arriving now at another viewing window. North. Through this northward-facing window, Langdon gazed down at the familiar silhouette of the White House directly in front of him. He raised his eyes to the horizon, where the straight line of Sixteenth Street ran due north toward the House of the Temple. I am due south of Heredom. He continued around the perimeter to the next window. Looking west, Langdon's eyes traced the long rectangle of the reflecting pool to the Lincoln Memorial, its classical Greek architecture inspired by the Parthenon in Athens, Temple to Athena–goddess of heroic undertakings. Annuit coeptis, Langdon thought. God favors our undertaking. Continuing to the final window, Langdon gazed southward across the dark waters of the Tidal Basin, where the Jefferson Memorial shone brightly in the night. The gently sloping cupola, Langdon knew, was modeled after the Pantheon, the original home to the great Roman gods of mythology. Having looked in all four directions, Langdon now thought about the aerial photos he had seen of the National Mall–her four arms outstretched from the Washington Monument toward the cardinal points of the compass. I am standing at the crossroads of America. Langdon continued back around to where Peter was standing. His mentor was beaming. â€Å"Well, Robert, this is it. The Lost Word. This is where it's buried. The Masonic Pyramid led us here.† Langdon did a double take. He had all but forgotten about the Lost Word. â€Å"Robert, I know of nobody more trustworthy than you. And after a night like tonight, I believe you deserve to know what this is all about. As promised in legend, the Lost Word is indeed buried at the bottom of a winding staircase.† He motioned to the mouth of the monument's long stairwell. Langdon had finally started to get his feet back under him, but now he was puzzled. Peter quickly reached into his pocket and pulled out a small object. â€Å"Do you remember this?† Langdon took the cube-shaped box that Peter had entrusted to him long ago. â€Å"Yes . . . but I'm afraid I didn't do a very good job of protecting it.† Solomon chuckled. â€Å"Perhaps the time had come for it to see the light of day.† Langdon eyed the stone cube, wondering why Peter had just handed it to him. â€Å"What does this look like to you?† Peter asked. Langdon eyed the 1514 and recalled his first impression when Katherine had unwrapped the package. â€Å"A cornerstone.† â€Å"Exactly,† Peter replied. â€Å"Now, there are a few things you might not know about cornerstones. First, the concept of laying a cornerstone comes from the Old Testament.† Langdon nodded. â€Å"The Book of Psalms.† â€Å"Correct. And a true cornerstone is always buried beneath the ground–symbolizing the building's initial step upward out of the earth toward the heavenly light.† Langdon glanced out at the Capitol, recalling that its cornerstone was buried so deep in the foundation that, to this day, excavations had been unable to find it. â€Å"And finally,† Solomon said, â€Å"like the stone box in your hand, many cornerstones are little vaults . . . and have hollow cavities so that they can hold buried treasures . . . talismans, if you will– symbols of hope for the future of the building about to be erected.† Langdon was well aware of this tradition, too. Even today, Masons laid cornerstones in which they sealed meaningful objects–time capsules, photos, proclamations, even the ashes of important people. â€Å"My purpose in telling you this,† Solomon said, glancing over at the stairwell, â€Å"should be clear.† â€Å"You think the Lost Word is buried in the cornerstone of the Washington Monument?† â€Å"I don't think, Robert. I know. The Lost Word was buried in the cornerstone of this monument on July 4, 1848, in a full Masonic ritual.† Langdon stared at him. â€Å"Our Masonic forefathers buried a word?!† Peter nodded. â€Å"They did indeed. They understood the true power of what they were burying.† All night, Langdon had been trying to wrap his mind around sprawling, ethereal concepts . . . the Ancient Mysteries, the Lost Word, the Secrets of the Ages. He wanted something solid, and despite Peter's claims that the key to it all was buried in a cornerstone 555 feet beneath him, Langdon was having a hard time accepting it. People study the mysteries for entire lifetimes and are still unable to access the power allegedly hidden there. Langdon flashed on Durer's Melencolia I–the image of the dejected Adept, surrounded by the tools of his failed efforts to unveil the mystical secrets of alchemy. If the secrets can actually be unlocked, they will not be found in one place! Any answer, Langdon had always believed, was spread across the world in thousands of volumes . . . encoded into writings of Pythagoras, Hermes, Heraclitus, Paracelsus, and hundreds of others. The answer was found in dusty, forgotten tomes on alchemy, mysticism, magic, and philosophy. The answer was hidden in the ancient library of Alexandria, the clay tablets of Sumer, and the hieroglyphs of Egypt. â€Å"Peter, I'm sorry,† Langdon said quietly, shaking his head. â€Å"To understand the Ancient Mysteries is a lifelong process. I can't imagine how the key could possibly rest within a single word.† Peter placed a hand on Langdon's shoulder. â€Å"Robert, the Lost Word is not a `word.'† He gave a sage smile. â€Å"We only call it the `Word' because that's what the ancients called it . . . in the beginning.† CHAPTER 130 In the beginning was the Word. Dean Galloway knelt at the Great Crossing of the National Cathedral and prayed for America. He prayed that his beloved country would soon come to grasp the true power of the Word–the recorded collection of the written wisdom of all the ancient masters–the spiritual truths taught by the great sages. History had blessed mankind with the wisest of teachers, profoundly enlightened souls whose understanding of the spiritual and mental mysteries exceeded all understanding. The precious words of these Adepts–Buddha, Jesus, Muhammad, Zoroaster, and countless others–had been transmitted through history in the oldest and most precious of vessels. Books. Every culture on earth had its own sacred book–its own Word–each one different and yet each one the same. For Christians, the Word was the Bible, for Muslims the Koran, for Jews the Torah, for Hindus the Vedas, and on and on it went. The Word shall light the way. For America's Masonic forefathers, the Word had been the Bible. And yet few people in history have understood its true message. Tonight, as Galloway knelt alone within the great cathedral, he placed his hands upon the Word–a well-worn copy of his own Masonic Bible. This treasured book, like all Masonic Bibles, contained the Old Testament, the New Testament, and a treasure trove of Masonic philosophical writings. Although Galloway's eyes could no longer read the text, he knew the preface by heart. Its glorious message had been read by millions of his brethren in countless languages around the world. The text read: TIME IS A RIVER . . . AND BOOKS ARE BOATS. MANY VOLUMES START DOWN THAT STREAM, ONLY TO BE WRECKED AND LOST BEYOND RECALL IN ITS SANDS. ONLY A FEW, A VERY FEW, ENDURE THE TESTINGS OF TIME AND LIVE TO BLESS THE AGES FOLLOWING. There is a reason these volumes survived, while others vanished. As a scholar of faith, Dean Galloway had always found it astonishing that the ancient spiritual texts–the most studied books on earth–were, in fact, the least understood. Concealed within those pages, there hides a wondrous secret. One day soon the light would dawn, and mankind would finally begin to grasp the simple, transformative truth of the ancient teachings . . . and take a quantum leap forward in understanding his own magnificent nature. CHAPTER 131 The winding staircase that descends the spine of the Washington Monument consists of 896 stone steps that spiral around an open elevator shaft. Langdon and Solomon were making their way down, Langdon still grappling with the startling fact that Peter had shared with him only moments ago: Robert, buried within the hollow cornerstone of this monument, our forefathers placed a single copy of the Word–the Bible–which waits in darkness at the foot of this staircase. As they descended, Peter suddenly stopped on a landing and swung his flashlight beam to illuminate a large stone medallion embedded in the wall. What in the world?! Langdon jumped when he saw the carving. The medallion depicted a frightening cloaked figure holding a scythe and kneeling beside an hourglass. The figure's arm was raised, and his index finger was extended, pointing directly at a large open Bible, as if to say: â€Å"The answer is in there!† Langdon stared at the carving and then turned to Peter. His mentor's eyes shone with mystery. â€Å"I'd like you to consider something, Robert.† His voice echoed down the empty stairwell. â€Å"Why do you think the Bible has survived thousands of years of tumultuous history? Why is it still here? Is it because its stories are such compelling reading? Of course not . . . but there is a reason. There is a reason Christian monks spend lifetimes attempting to decipher the Bible. There is a reason that Jewish mystics and Kabbalists pore over the Old Testament. And that reason, Robert, is that there exist powerful secrets hidden in the pages of this ancient book . . . a vast collection of untapped wisdom waiting to be unveiled.† Langdon was no stranger to the theory that the Scriptures contained a hidden layer of meaning, a concealed message that was veiled in allegory, symbolism, and parable. â€Å"The prophets warn us,† Peter continued, â€Å"that the language used to share their secret mysteries is a cryptic one. The Gospel of Mark tells us, `Unto you is given to know the mystery . . . but it will be told in parable.' Proverbs cautions that the sayings of the wise are `riddles,' while Corinthians talks of `hidden wisdom.' The Gospel of John forewarns: `I will speak to you in parable . . . and use dark sayings.' â€Å" Dark sayings, Langdon mused, knowing this strange phrase made numerous odd appearances in Proverbs as well as in Psalm 78. I will open my mouth in a parable and utter dark sayings of old. The concept of a â€Å"dark saying,† Langdon had learned, did not mean that the saying was â€Å"evil† but rather that its true meaning was shadowed or obscured from the light. â€Å"And if you have any doubts,† Peter added, â€Å"Corinthians overtly tells us that the parables have two layers of meaning: `milk for babes and meat for men'–where the milk is a watered-down reading for infantile minds, and the meat is the true message, accessible only to mature minds.† Peter raised the flashlight, again illuminating the carving of the cloaked figure pointing intently at the Bible. â€Å"I know you are a skeptic, Robert, but consider this. If the Bible does not contain hidden meaning, then why have so many of history's finest minds–including brilliant scientists at the Royal Society–become so obsessed with studying it? Sir Isaac Newton wrote more than a million words attempting to decipher the true meaning of the Scripture, including a 1704 manuscript that claimed he had extracted hidden scientific information from the Bible!† Langdon knew this was true. â€Å"And Sir Francis Bacon,† Peter continued, â€Å"the luminary hired by King James to literally create the authorized King James Bible, became so utterly convinced that the Bible contained cryptic meaning that he wrote in his own codes, which are still studied today! Of course, as you know, Bacon was a Rosicrucian and penned The Wisdom of the Ancients.† Peter smiled. â€Å"Even the iconoclastic poet William Blake hinted that we should read between the lines.† Langdon was familiar with the verse: BOTH READ THE BIBLE DAY AND NIGHT, BUT THOU READ BLACK WHERE I READ WHITE. â€Å"And it wasn't just the European luminaries,† Peter continued, descending faster now. â€Å"It was here, Robert, at the very core of this young American nation, that our brightest forefathers–John Adams, Ben Franklin, Thomas Paine–all warned of the profound dangers of interpreting the Bible literally. In fact, Thomas Jefferson was so convinced the Bible's true message was hidden that he literally cut up the pages and reedited the book, attempting, in his words, `to do away with the artificial scaffolding and restore the genuine doctrines.' â€Å" Langdon was well aware of this strange fact. The Jeffersonian Bible was still in print today and included many of his controversial revisions, among them the removal of the virgin birth and the resurrection. Incredibly, the Jeffersonian Bible had been presented to every incoming member of Congress during the first half of the nineteenth century. â€Å"Peter, you know I find this topic fascinating, and I can understand that it might be tempting for bright minds to imagine the Scriptures contain hidden meaning, but it makes no logical sense to me. Any skilled professor will tell you that teaching is never done in code.† â€Å"I'm sorry?† â€Å"Teachers teach, Peter. We speak openly. Why would the prophets–the greatest teachers in history–obscure their language? If they hoped to change the world, why would they speak in code? Why not speak plainly so the world could understand?† Peter glanced back over his shoulder as he descended, looking surprised by the question. â€Å"Robert, the Bible does not talk openly for the same reason the Ancient Mystery Schools were kept hidden . . . for the same reason the neophytes had to be initiated before learning the secret teachings of the ages . . . for the same reason the scientists in the Invisible College refused to share their knowledge with others. This information is powerful, Robert. The Ancient Mysteries cannot be shouted from the rooftops. The mysteries are a flaming torch, which, in the hands of a master, can light the way, but which, in the hands of a madman, can scorch the earth.† Langdon stopped short. What is he saying? â€Å"Peter, I'm talking about the Bible. Why are you talking about the Ancient Mysteries?† Peter turned. â€Å"Robert, don't you see? The Ancient Mysteries and the Bible are the same thing.† Langdon stared in bewilderment. Peter was silent for several seconds, waiting for the concept to soak in. â€Å"The Bible is one of the books through which the mysteries have been passed down through history. Its pages are desperately trying to tell us the secret. Don't you understand? The `dark sayings' in the Bible are the whispers of the ancients, quietly sharing with us all of their secret wisdom.† Langdon said nothing. The Ancient Mysteries, as he understood them, were a kind of instruction manual for harnessing the latent power of the human mind . . . a recipe for personal apotheosis. He had never been able to accept the power of the mysteries, and certainly the notion that the Bible was somehow hiding a key to these mysteries was an impossible stretch. â€Å"Peter, the Bible and the Ancient Mysteries are total opposites. The mysteries are all about the god within you . . . man as god. The Bible is all about the God above you . . . and man as a powerless sinner.† â€Å"Yes! Exactly! You've put your finger on the precise problem! The moment mankind separated himself from God, the true meaning of the Word was lost. The voices of the ancient masters have now been drowned out, lost in the chaotic din of self-proclaimed practitioners shouting that they alone understand the Word . . . that the Word is written in their language and none other.† Peter continued down the stairs. â€Å"Robert, you and I both know that the ancients would be horrified if they saw how their teachings have been perverted . . . how religion has established itself as a tollbooth to heaven . . . how warriors march into battle believing God favors their cause. We've lost the Word, and yet its true meaning is still within reach, right before our eyes. It exists in all the enduring texts, from the Bible to the Bhagavad Gita to the Koran and beyond. All of these texts are revered upon the altars of Freemasonry because Masons understand what the world seems to have forgotten . . . that each of these texts, in its own way, is quietly whispering the exact same message.† Peter's voice welled with emotion. † `Know ye not that ye are gods?'† Langdon was struck by the way this famous ancient saying kept surfacing tonight. He had reflected on it while talking to Galloway and also at the Capitol Building while trying to explain The Apotheosis of Washington. Peter lowered his voice to a whisper. â€Å"The Buddha said, `You are God yourself.' Jesus taught that `the kingdom of God is within you' and even promised us, `The works I do, you can do . . . and greater.' Even the first antipope–Hippolytus of Rome–quoted the same message, first uttered by the gnostic teacher Monoimus: `Abandon the search for God . . . instead, take yourself as the starting place.' † Langdon flashed on the House of the Temple, where the Masonic Tyler's chair bore two words of guidance carved across its back: KNOW THYSELF. â€Å"A wise man once told me,† Peter said, his voice faint now, â€Å"the only difference between you and God is that you have forgotten you are divine.† â€Å"Peter, I hear you–I do. And I'd love to believe we are gods, but I see no gods walking our earth. I see no superhumans.You can point to the alleged miracles of the Bible, or any other religious text, but they are nothing but old stories fabricated by man and then exaggerated over time.† â€Å"Perhaps,† Peter said. â€Å"Or perhaps we simply need our science to catch up with the wisdom of the ancients.† He paused. â€Å"Funny thing is . . . I believe Katherine's research may be poised to do just that.† Langdon suddenly remembered that Katherine had dashed off from the House of the Temple earlier. â€Å"Hey, where did she go, anyway?† â€Å"She'll be here shortly,† Peter said, grinning. â€Å"She went to confirm a wonderful bit of good fortune.† Outside, at the base of the monument, Peter Solomon felt invigorated as he inhaled the cold night air. He watched in amusement as Langdon stared intently at the ground, scratching his head and looking around at the foot of the obelisk. â€Å"Professor,† Peter joked, â€Å"the cornerstone that contains the Bible is underground. You can't actually access the book, but I assure you it's there.† â€Å"I believe you,† Langdon said, appearing lost in thought. â€Å"It's just . . . I noticed something.† Langdon stepped back now and surveyed the giant plaza on which the Washington Monument stood. The circular concourse was made entirely of white stone . . . except for two decorative courses of dark stone, which formed two concentric circles around the monument. â€Å"A circle within a circle,† Langdon said. â€Å"I never realized the Washington Monument stands at the center of a circle within a circle.† Peter had to laugh. He misses nothing. â€Å"Yes, the great circumpunct . . . the universal symbol for God . . . at the crossroads of America.† He gave a coy shrug. â€Å"I'm sure it's just a coincidence.† Langdon seemed far off, gazing skyward now, his eyes ascending the illuminated spire, which shone stark white against the black winter sky. Peter sensed Langdon was beginning to see this creation for what it truly was . . . a silent reminder of ancient wisdom . . . an icon of enlightened man at the heart of a great nation. Even though Peter could not see the tiny aluminum tip at the top, he knew it was there, man's enlightened mind straining toward heaven. Laus Deo. â€Å"Peter?† Langdon approached, looking like a man who'd endured some kind of mystical initiation. â€Å"I almost forgot,† he said, reaching into his pocket and producing Peter's gold Masonic ring. â€Å"I've been wanting to return this to you all night.† â€Å"Thank you, Robert.† Peter held out his left hand and took the ring, admiring it. â€Å"You know, all the secrecy and mystery surrounding this ring and the Masonic Pyramid . . . it had an enormous effect on my life. When I was a young man, the pyramid was given to me with the promise that it hid mystical secrets. Its mere existence made me believe there were great mysteries in the world. It piqued my curiosity, fueled my sense of wonder, and inspired me to open my mind to the Ancient Mysteries.† He smiled quietly and slipped the ring into his pocket. â€Å"I now realize that the Masonic Pyramid's true purpose was not to reveal the answers, but rather to inspire a fascination with them.† The two men stood in silence for a long while at the foot of the monument. When Langdon finally spoke, his tone was serious. â€Å"I need to ask you a favor, Peter . . . as a friend.† â€Å"Of course. Anything.† Langdon made his request . . . firmly. Solomon nodded, knowing he was right. â€Å"I will.† â€Å"Right away,† Langdon added, motioning to the waiting Escalade. â€Å"Okay . . . but one caveat.† Langdon rolled his eyes, chuckling. â€Å"Somehow you always get the last word.† â€Å"Yes, and there is one final thing I want you and Katherine to see.† â€Å"At this hour?† Langdon checked his watch. Solomon smiled warmly at his old friend. â€Å"It is Washington's most spectacular treasure . . . and something very, very few people have ever seen.†

European Influence on Japan & China Essay

The European arrival had an effect on both China and Japan. China and Japan had some similar and different reactions to the arrival of the Europeans. In China and Japan, the European arrival affected the technologies and economies of both societies similarly, whereas the reason of isolation differed in these societies. The technology of the European’s following their arrival penetrated both the Japanese and Chinese societies. In China, in order to gain the elites interest in the Christian religion, the Jesuit missionaries introduced the technology of cannons and clocks. In Japan during the 1950s, the Portuguese technology of clock and gun making, influenced society strongly. The Japanese and Chinese both openly accepted the new European technologies because they felt they needed to accept the technologies to keep up with the Europeans, who seemed more technologically advanced. In both China and Japan, people were being converted to Christianity. They were being converted from top to bottom. Once a year, China and Japan, were able to trade with the Europeans. They isolated themselves and traded with each other. Japan and China also had some different reactions to the European arrival. They had different reasons for isolating. After Zheng He died, the Chinese government decided to close the ports, except two. The Chinese and the Europeans exchanged goods once a year for the next 400 years. China agreed with the isolation because the scholar gentry saw the voyages as a waste of resources. They rather spend the money on defending China. Japan grew doubts with the European intentions, that both merchants and missionaries might subvert the existing social order. This led to official measures to restrict foreign activities in Japan. Under Ieyasu and his successors, the persecution of the Christians increased to isolate Japan from outside influences. In the 1630s, all the Japanese ships were forbidden to trade or even sail overseas. By the mid-17th century, Japan’s retreat into almost total isolation was complete. A major difference was after Japan isolated themselves, an elite group still remained interested in the Europeans. Unlike the Chinese who looked down on the Europeans and ignored what they had to offer and stay isolated with them. The Chinese and Japanese had similar and different reactions to the arrival of the Europeans. Both of these societies had a technology advancement. Also, they both isolated themselves from the Europeans and instead they traded with each other. They had different reasons for isolating. Also, an important difference was that in Japan, a group remained interested in the Europeans. This differed from the Chinese because after they isolated, they didn’t want anything to do with the Europeans.

Mccain

External Business environmental challenges on the business One of the biggest problems of McCain was obesity In children. This problem related to external challenges, which faced by the organization. McCain had to focused on the healthy problem occurred due to their products. McCain had to understand the dietary of the customers, which should be balanced as well as healthy. In relation to understand the customer's requirement and other issues, it was mandatory for the company to analyses its environment whether it was internal or external.Following figure show the external challenges: (http://yeomanry. Mom, 2014) After doing the slept analysis organization needs to examine those factors, which affects the business. When slept has been occurred then, company has to made changes according to the environment changes. Some external challenges is as below: Social Factors: It Is one of the Important factors that affects directly to the business. Consumer behavior depends resolute by the pr esent trend. Habits Like eating, drinking changes with time to time. McCain sales growth declined due to some healthy eating patterns of the consumers.McCain reduces the quantity of salt in their potato products. Mccain claimed that their potato products are lower in fats and saturated fats. McCain also promote its product to the public to increase the sales. They communicate with people through campaigns. McCain also made strong public relation with the help of advertisement. Legal Factors: Every company has to deal with the legal criteria set by the government. There must be some standard that are set by the company to meet with the government rules and regulations.Government set standards on the behalf of consumers. They made various traffic lights labels which shows the quality of the product that help the nonusers to choose the right product. For example red dot represent high level of ingredients like fat and unsaturated fats and green dot represent low level of fat. There are some guidelines set by the government for the companies in relation of quality. Some of the guidelines are also called daily amount guidelines, which represent the daily intake of the fats and unsaturated fats.Advertising also comes under legal factors. It supervised by the voluntary body that related to advertising industry. This special authority called advertising standards authority. Economic Factors: One of the mall challenges of the company Is people's rolling Income. This was big problem for the company because as the Income rises then, people prefer to buy the superior varlets of the products. As the people becoming richer they prefer ready to eat meal and ready meals rather than buying potatoes and chips at home. So responsible for health.Then, McCain introduces the various products like Mccain straight cut, oven chips, Home fries, roast potatoes etc. Technological factors: Technology is the most important challenges for the company. Food technology plays an important role in this modern economy. Some of the challenges for the companies like frozen products, muesli bars etc. One of the main challenges for the company how to retain the quality of the potatoes products. B. Two strength and two Weaknesses of McCain foods in emerging the organization sustainability. Strength McCain company toughly emphases on the quality of its products.It hires more than 90 agronomist's potato experts at 55 production facilities across 6 continents, to help local producers to grow best quality of potatoes and vegetables that the company can use those products in manufacturing its products and also confirm consistent supply. McCain Research Farms concentrate on emerging quality agricultural products. (An, 2014) McCain Company drives a Potato Processing Technology Centre in Canada and frequently improvisation's manufacturing processes in order to increase efficiency and product value. An, 2014) The company's thoughtlessly on keeping high quality levels for raw materials d istinguishes its products from others intone market and increases the brand image. (An, 2014) Nakedness's The company has slightest presence in developing the organic segments of food. Organic and natural food products are now becoming the rapid growing classification in food retail sector. Consumer awareness organic alternatives for food products and increasing health anxieties about Junk food have headed to chirography in this particular segment. An, 2014) Restricted existence in a reckless growing product sector mayhap an unfavorable mark on the company's market share. Downtrends status McCain has been popular as a private company; it could face problems in getting money for outgrowth owing to its ownership status. Private ownership's the company at a reasonable disadvantage. (An, 2014) (Strauss, 2014) Adaptability culture is the most corporate culture that McCain need to adopt for their tragedies. In adaptability culture, company develops in external environment. Company need to response as soon as possible when the company makes the decision.It involves the high-risk decisions that get quick feedback from the environment. Managers need to encourage the company staff so that company needs to detect rapidly. Three reasons that make adaptability culture better than others are as below: 1 . The first reason is that it is flexible by nature. Whenever, company need to change the strategies according to customers needs then, company can change any time and very easily. Customer need changes according to time then, it is the first duty of the company to be focused the needs of customer. 2.Employees have self-rule to make decision and also act freely to meet the new needs of customers. Employees should know regarding their decision. If the employees participate a lot in the responsiveness to customers then, those decision will be highly value and appreciated. 3. Company need to be creative as well doing experimentation on new products according to the customers ne ed. For example, In the market, there is customer need come in front of the company and company start working on it then, many can easily introduce the new product.Vision & Strategy (http://www. McCain. Com, 2014) Some of the company objectives are as below: Acquire the highest market share Boost the sales of the company Satisfy the customer and its need Personalization for shareholders Growing together business strategy customers and consumers through the development of our people, stewardship of the environment and our actions as a responsible corporate citizen We believe that social responsibility is an expression of how we conduct all aspects of our business and cannot be defined within a single program or policy.This belief is reflected in the Growing Together business strategy we introduced in 2005, which is anchored by three interdependent pillars: Distributed and networked leadership: Developing people as leaders at all levels of the organization and collaborating through le arning and project networks to leverage our global scale and best practices. Market facing: Making decisions at the closest level to the customer and creating demand for our products through innovation, value, differentiation and in the way we interact with our customers and consumers. Global CARS report, 2009) Corporate social responsibility: Making good, nutritious food on the principles of sustainability and food safety and supporting our employees, customers and consumers in their aspiration for a Better Life. Coach section of this report – Respecting our Environment, Inspiring Wellness and Positively Impacting our People – describes the policies, practices and initiatives that underpin our strategy and the progress we are making on our Journey of continuous improvement in advancing our goals in a socially responsible manner. Global CARS report, 2009) Advantages and disadvantages of Management-by-objective Management by Objectives MOB can likewise be alluded as Man agement by Results or Goal Management, and is focused around the presumption that contribution prompts responsibility and if a worker takes part in objective setting and additionally setting norms for estimation of execution towards that objective, then the representative will be roused to perform better and in a way that straightforwardly helps the accomplishment of authoritative goals. Advantages of Management by Objectives 1 .Since Management by targets (MOB) is a result-turned process and concentrates n setting and controlling objectives, if urges chiefs to do unity gritty arranging. 2. Both the top managers and the Juniors realize what is anticipated from them and consequently there is no part imprecision or disarray. When both top managers and subordinates know each other about there work then, there will be no confusion between them. Disadvantages of Management by Objectives 1 . MOB can be accomplishing because it has the complete backing of the top management.If the McCain t op manager will not supportive with there staff then, the strategies will no completed. Whatever, strategic plan made by the management should be related to staff. 2. They may be less weight to co-occur with the administration during setting objectives and goals and these objectives may be set unreasoningly high. This may low down their assurance and they may get to be suspicious about the logic behind MOB. Control System to improve Production process and product quality Controlling is one of the most important functions, which consist planning, organizing, staffing and directing.It is important because It help the company to correct the errors and to take corrective action so that divergence from tankards are minimized and the set goals of the company are achieved in the desired manner. Control in management means to improve the production process, Feed forward Control Concurrent Control Feedback Control Afterwards Afterwards is a communication term that refers to regulate impact t o a person or a company from which anybody can expect output. Feed Forward is not like pre- feedback, because it helps to measure an output and also help in sending the feedback.For example, if pre-feedback is occurred during quality and production process which is really easy for the organization to get an idea of the output. It also involves a document of review and giving post information regarding the production and quality process. Concurrent control will be control that happens in the meantime as a task is happening. This observing and controlling comprises of the courses of action that project execution so that potential issues might be distinguished in a promising way and correct procedures could be made, when needed, to control the execution of the task.If any project is under process in production unit then, concurrent control strategy can be used during that process. It will help the managers to observe and intro and of course, actions can be taken for correct execution o f the process. Feedback is a method in which data about the past or the present impacts the same phenomenon in the present or future. As a part of a chain of its effect and causes that forms a circuit, the occasion is said to â€Å"feedback† into itself. Feedback† exists between two sections when each affects the other. As a company tries to improve its performance, feedback helps to make some adjustments. Feedback helps to motivate the people in the work place. When anybody receives positive or negative feedback, hat person decides how to apply that feedback on his or her Job. In the production and quality process of the company, if any employee receive the positive feedback then, that person will automatically motivated towards company.

International Trade 1 Essay Example | Topics and Well Written Essays - 1500 words

International Trade 1 - Essay Example United States Department of Agriculture’s Economic Research Service (USDA’s ERS) report says that during the period 2000 to 2004 the world economy grew from $39190 billion to $43363 billion. (www.ers.usda.gov). WTO report says that the Asian region recorded an export growth of 14.5 in 2004. Real merchandise imports in South America grew by 18.5 per cent, which was twice as fast as world trade in 2004. Africa’s trade expanded strongly in 2004. North America’s export recovery, which started in 2003, gained momentum in 2004. The enlargement process of the European Union towards the east fostered an integration process between central and Eastern Europe resulting in sharp rise in intra – industry exchanges, e.g. automobiles. The GDP growth of Asia more than doubled from 2.37 percent in 2001 to 5.42 percent in 2004. Thus, growth of an economy automatically increases the economic components like exports and trade The major chunk of merchandise trading, after oil, around the globe happens in iron and steel, ores, non-ferrous metals and fuels. The growth of this sector also contributes to the growth of global trade. Historically the US has been the major importer of iron and steel, ores and non-ferrous metals. The growth in real estate and construction industry fuelled this need for iron and steel. Being the largest manufacturer of goods also made the US the largest consumer of non-ferrous metals like copper and aluminium. With the emergence of China, the consumption of non-ferrous base metals has gone up drastically. Chinese imports of the metals have increased to extent, where the demand has overshot the production. This increased the prices of base metals like copper. The price of copper in the London Exchange in April 2000 was around $ 1600 per metric tonne and in April 2004 it was around $2200 per metric

Dissertation proposal (The Level of Interest within Generation Y Essay

Dissertation proposal (The Level of Interest within Generation Y towards Social network Marketing) - Essay Example Background of Research Questions Social Network Marketing has gained prominence in the last decade as more and more companies are marketing primarily through social networking sites. The main purpose of social networking sites is to connect users with one another. As the internet age blooms with every passing year, social networking has become the most widely used communication tool by users around the world. ‘Compete.com (2008), a Web traffic analysis company, reported that the largest online social networking site (as of November 2008) was MySpace, with 56 million unique visitors per month, closely followed by Facebook, with 49 million unique visitors’ (Trusov, Bucklin and Pauwels, 2009). This addiction towards social networking presents marketers with a good opportunity of reaching towards consumers. Through social networks, brands develop a personality and provide a forum for the users to interact with the brand, thus making way for a more informal form of marketing. With the saturation in market that most industries are now facing, it becomes increasing hard to capture the attention of the consumers who have become cynical towards advertising and marketing in general. Social networking sites allow consumers to share their opinions, ideas and reviews about products and services. On account of this, social networking sites support word-of-mouth, which is the most effective form of advertising and specifically for the younger generation. With advertisers taking full use of their creative license, consumers have become wary of products advertising impossibility shiny long hair and a perfect social life. They simply do not believe these advertisements. This research will aim to uncover the connection that Generation Y has with social networking sites. With the advertising world changing at a very fast pace, it is important that marketers are able to connect with the Generation Y through their preferred platform. This research provides a way forward to marketers. Literature Review ‘Today’s Generation Y, encompassing individuals born between 1980 and 1995, represents an era of teens and young adults who grew up with technology and are among the most avid users of social networking’ (Rutledge, 2008) . Social Networking sites have become part of the life of Generation Y. As important as it is to have residential addresses, Generation Y considers it equally important to have a social networking address. Given the popularity of social networking sites among Generation Y, organizations have begun exploring advertising on these sites. ‘Social Networking is just one of the many consumer technologies, including blogs, wikis and virtual worlds, to cross over to the corporate world’ (Smith and Salvendy, 2009). But the advertising content must differ since Generation Y has become used to the traditional advertising techniques. They tend to tune out the advertising of women falling around men with just one spray of a perfume. To reach Generation Y, organizations have to more honest and direct in their advertising techniques (Garland, 2007). Generation Y is more of a rebel and has shaped their opinions and views around the opinions of views of their own peers. Parents have not been the guiding figures in the lives of Generation Y since they were raised in a time when both parents

Company going through a change process (Crave Outsourcing) - Apply Literature review

Company going through a change process (Crave Outsourcing) - Apply change theory - Literature review Example The present study would try to analyse the change management process of Crave Outsourcing with regards to its change in service delivery from IT, recruitment and accountancy to project management. The study would largely include the objectives of the firm that would help in initiating the change process. Apart from this the study would also include an analysis of the existing literature towards analysis the theoretical framework of change management. In addition to the literature review the problem solving techniques would be focussed upon which would include the six thinking hats strategy. Finally a set of plausible recommendations would be framed that would enable Crave Outsourcing to generate sustainable competitive advantage in the business environment. Objectives The main objectives of the change management process of Crave Outsourcing would largely include the following aspects. ... ms to bring greater accountability and maintaining sustainability The objectives stated above would largely help in ensuring better sustainability and would also help the firm to diversify its service offering that would help the firm to venture into business segments that can generate greater profitability for the firm in the long run. The fulfilment of the objectives would also enable the firm to move into ventures that are more profitable which would help in ensuring better prospects for the organization in the future. Company Snapshot Crave Outsourcing operates from its head office based in London top generate a plethora of consulting services pertaining to the domain of technology, human resources functions like recruiting the right individual and other financial and accounting functions. The consulting and outsourcing firm operating from United Kingdom gains wide amount of recognition by opening up a number of branches in several regions around the world. In respect to client s ervicing the company aims to render services of better quality by making maximum optimisation of the existing resources. The company through its operation in various other regions strategizes to gain the view of local markets and customer base by recruiting local people. These local set of employees help the company management to gain a wider understanding of the local environments to be able to deliver enhanced services and thereby augmenting the goodwill. Then client base of the company is mainly constituted by small and medium sized enterprises located mainly in regions like United Kingdom, Nigeria and other global regions (Crave Outsourcing, 2009; Crave Outsourcing-c 2009). Literature Review Change management is something that tests every manager’s mettle. When an enterprise decides

Social science research Assignment Example | Topics and Well Written Essays - 2000 words

Social science research - Assignment Example Apparently both methods draw considerable advantages and disadvantages and in this essay, I seek to critically evaluate them and on their application in social science works. The main advantage of the qualitative methodology is that, it employs a descriptive, modest, and inductive techniques of data collection that are compatible with social science works (McRoy, n.d.). It is the most advocated method when social workers are studying issues or topics that do not agree with the quantitative research approach. From a social psychological basis, qualitative method is coherent with person-in-environment of social science professional practice. In dealing with patient’s issues and treatment, doctors, nurses, and clinicians rely on interviews to gather information about a particular client’s illness. Such practitioners, therefore, follow a series of intuitions and working assumptions that are based on observations made through ongoing interaction with the patients. Most training offered to social science practitioner is based on qualitative research, whereby from a qualitative view, they are trained to observe at each case individually (McRoy, n. d.). Unlike quantitative method that focuses on broader social sciences topics; qualitative approach has the advantage of addressing micro-level studies. The micro-level issues include those of small groups, subjects, or organizations and research that are not so much physical, but rather needs a psychological approach. When qualitative research is applied in such topics, it draws a wide range of facets that depict a picture of wholeness in terms of group(s), person (s) or settings of the situation. The depicted picture of a study by a qualitative researcher on micro-level topics is holistic because it pulls all the necessary colours, structures, and textures into the painting(Tewksbury, 2009).The after analysis result is plainly contrasting and can never

Examine the three theoretical models of opportunity recognition Essay

Examine the three theoretical models of opportunity recognition - Essay Example In our second section, we de-construct the factors that impact opportunity recognition. Here, we explain the various ways in which cognition impacts opportunity recognition. In our third section, we study the influence of human and social capital on the opportunity recognition process. In our fourth and last section, we summarize the arguments and provide concluding remarks. I. Opportunity Recognition: theoretical constructs The capacity to identify and spot a successful business opportunity is the hallmark of a preliminary stage in any new entrepreneurial venture creation (Ardichvili, Cardozo & Ray 2003). Once an opportunity is recognized, the individual then proceeds to analyze it through different angles. What distinguishes an individual’s capability to identify the right opportunity? This forms the basis of the discussion for our paper. When we extend the unit of analysis from an individual to a firm, then we can also infer that opportunity recognition follows from a caref ul, analytical process (Denrell, Fang & Winter 2003). Possibly, we could conclude that an individual may not possess all the resources that are at the disposal of a firm, yet an individual develops his or her own methods to evaluate a business opportunity. ... These form the broad backbone of the opportunity recognition process. Shane (2003 p.60) also lists out three factors that influence the possibility of gaining information access: 1) prior life and work experience, 2) the social network structure and 3) information search. Previous experiences in a particular field helps people gain awareness and confidence about the domain. Sometimes, this translates into a deeper capacity for identifying unexplored business possibilities within the chosen business realm (Bishop 2011). Again, if the individuals belong to a family that has predominantly focused on their own businesses, this could influence them to spot business opportunities more easily. In essence, family occupation could influence individuals’ attitude and thought processes about business. Certain functional domains could also guide individuals towards entrepreneurial domains. For examples, people with more exposure to sales and marketing tend to have a good grasp of the mark et. They understand consumer behavior to some extent and can appreciate the process flow of business. This could be one factor that gives them an entrepreneurial spirit. Membership of a network also assists in the opportunity recognition process. Ozgen & Baron (2007) suggest that individuals may gain information about new business ventures from their mentors, business networks and professional associations. For individuals who have been entrepreneurs before, this would help them identify new business ideas more easily (Ucbasaran, Westhead & Wright 2009). Networks could be formal ones such as that of colleagues sharing the same work function at the employer’s premises. Another possibility is a group that meets informally after

Math problem Essay Example | Topics and Well Written Essays - 750 words

Math problem - Essay Example This means that there is some question as to whether this sample is sufficiently random, and whether that sample accurately represents the population. Another question to ask is how representative the sample was, by having only 50 represented could result to sample size as reported in guideline 3 of the article. In addition, the data that is used comes from the parents, rather than a single observer. As a result, there is likely to be significant variation in the observed results. Lastly, the question one remains asking is that the conclusions made are based on what? The study does not tell us the values of comparison in order to reach at the conclusion, it is important to consider guideline 8 in making the conclusions since sometimes the comparison values could not be significant to warrant such a conclusion made. The data reported by the parents is essentially anecdotal, rather than being an actual measure of the childs activity or energy level. The parents perception of how active their child is at any particular moment may be affected by the parents own mood and tolerance for activity at the time. If a parent is feeling particularly harried or overwhelmed, a lesser level of activity on the part of their child may seem more "active" than usual. Conversely, if a parent is feeling less stressed, they may be more tolerant of activity before they reach the point where the activity of the child seems "high" or "excessive". In other words, the parents perception of the childs activity may be inconsistent (According to guideline 1 we would conclude that the type of study is not appropriate), both due to the lack of a standard scale of measurement, and by their own emotional state. Finally, this particular study was sponsored by a group that has a vested interest in the outcome. In particular, the stated outcome favors the company that sponsored the study-this is similar to what we observe in guideline 2 of the article. This calls

Listening to children and young people Essay Example | Topics and Well Written Essays - 2500 words

Listening to children and young people - Essay Example Children who are capable of forming their own views have a right to receive and make known information they have through being attentively listened to, to effectively express their opinions, and to have those opinions taken into account in whatever matters affecting them. All views of the children ought to be given due weight in accordance to the age, maturity and capability of the children. According to Committee on the Rights of the Child (2009), all children with special education requirements are supposed to have a say in the roles of decision making in regards to their special education needs. Listening to the children and young ones do have very important concepts to be considered. Whoever interacts with children and young people should actually consider ensuring that they are able to benefit by growing up or developing uprightly. Through listening, adults are able to emphasize well with children by understanding the children’s inimitable thoughts, ideas, and feelings. Hence, there is creation of a child’s focused culture on communication. Effectively engaging with children and young people is very essential to Child, Youth and Family (CYF)’s task in supporting all families and adequately protecting children. By being in a position to understand the perspective of any child or young person is important in completing the assessment and development interventions. Demonstration of genuine interest in children or young people’s viewpoint, having a thorough understanding of the child/ young people’s personal development, and appreciating the environmental conditions which aid good communication with children, are so fundamental in genuine consultation and active inclusion of children in the decision making process. By ensuring that one is able to listen and try to understand children and the young people, he/she can help the child to develop the perceptions that they can be have some trust on you and come to you in case of anything to

Religious and Ethnic Diversity Paper Essay Example for Free

Religious and Ethnic Diversity Paper Essay The religious group that I have chosen is Hinduism and the ethnic/racial group that is not my own that I have chosen is Black (African Descent). I have chosen these two to explain how they are similar and how they are different. Hinduism is a religion that most people know nothing about. They are one of the world’s oldest religions as it has no beginning and precedes recorded history. Hinduism is a religion that leads the individual to personally experience the truth within themselves and reach full consciousness where man and God are one. After reading I have found that Hinduism differs from other religious groups because they believe in reincarnation whereas the other religious groups believe that once a person dies they either will go to heaven or hell depending on how they were when they were alive. The experience of Hinduism and others that do not share their beliefs or practices is criticism. They are often judged and criticized because they fail to distinguish between revelations. This religious group has contributed to American culture because they don’t believe in violence. They also contributed to the International Society for Krishna Consciousness. Some examples of prejudice and discrimination against Hinduism would be the Caste system or saying that they all burn their wives as well as discriminate against India where most come from. After reading the information to write this paper I must say it has helped me to understand this religion because I didn’t know that they were so much criticized. From what I read it seems like a very peaceful religion. They believe in reincarnation which I believe is possible, I mean, â€Å"why does a person have to go to heaven or hell†? I think that their religion is against violence which we definitely could have less of. The racial group that I have chosen was Black of African descent. This group differs from others because they have gone through so much over the years. They have experienced slavery which many other religions haven’t as they more so experienced discrimination. Blacks have not had such a great experience with other races because of always being mistreated because of their color. I think that they were brought over for slavery and still experience this even now these days from certain types of other races. Blacks have contributed to American culture though business and through entertainment. Most of the entertainment business is African American as well as sports and now we even have a black president. All very good contributions to American culture. Some examples of discrimination would be things like unequal pay for blacks performing the same work as whites, isolating blacks to only work in certain areas or specific locations, routinely denying black’s promotions, laying off blacks while whites keep their jobs and take on theirs as well. All of these are prime examples of discrimination that blacks go through in the United States. The sources of this discrimination would be whites or other races that were not black. Most of the cases that I read about though were whites still discriminating against black even after all the movements to move past racism. What I learned about this racial group is that even after all these years they are still faced with discrimination more than other racial groups. I can’t really say that I have learned any more than I already knew just because I see this happen regularly. I have a black/Cuban fiance and I have seen jobs do these things to him and now we have 2 boys that are mixed with white and black and have seen them faced with discrimination just because of the color of their skin. In conclusion, I believe that Hinduism and Black (African descent) both experience similar discrimination if the fact that they are perceived by first impressions and by the color of their skin. They are not given the luxury of being spoken with or getting to know them without the preconceived notion of their skin color. Both Hinduism and Blacks contribute to American culture business wise and yet many fail to know that. I can’t really say they are different because these two groups both experience prejudice and discrimination quite often. They are both groups of people that have done nothing wrong and just want to be a part of the world. References Nine Beliefs of Hinduism. (2013). Retrieved from http://www. himalayanacademy. com/readlearn/basics/nine-beliefs Hinduism and the Encounter with Other Faiths. (2008). Retrieved from http://www. worlddialogue. org/content. php? id=59 The Black Factor. (n. d. ). Retrieved from http://theblackfactor. blogspot. com/2008/03/examples-of-discrimination-harassment. html.

The Globe Construction Company Essay Example for Free

The Globe Construction Company Essay THE GLOBE CONSTRUCTION COMPANY The Globe Construction Company top management held a management workshop one weekend in April of 1989 for the purpose of discussing present policies and procedures with the ultimate objective of formalizing these in a new company operations manual. The company was established in the mid-50s by Mr. Eduardo Concepcion. The company grew at a very fast rate due to the construction boom in the government sector during the administration of ex-President Ferdinand E. Marcos, During the seventies and early eighties, the company constructed large projects for both the government and private sectors. However, most of its projects were for the government. Globe enjoyed high margins from its government projects during this period. Competition was not a problem. When Mrs. Aquino became President, private sector construction projects in the country outnumbered government projects. Competition became more stiff forcing Globe to reduce its margins when bidding for both sectors. This change in the environment led management to call the management workshop to discuss ways of improving organizational efficiency and effectiveness. The workshop was attended by the president, the vice-president for administration, the vice-president for operations, the purchasing manager and all the project managers ( See exhibit A for Organizational Chart) Project managers are the engineers in-charge of construction projects. The company employs twenty project managers. At present, there are about eighteen on-going projects at an estimated Bid price of at least P100 million each ranging from office condominiums, shopping centres and government buildings. The purchasing unit is headed by a purchasing manager to whom buyers report. Each buyer is completely in-charge of purchasing materials required by one or more construction projects. The VP for operations is over-all in-charge of on-going projects. During the workshop, one of the project managers, Isidro Remigio, proposed that the buyers should be located at the job site reporting to the Project Manager (See Exhibit B for the Organizational Chart at the Project Site) Mr. Remigio argued: â€Å"It takes so long, sometimes about 2 weeks, before Purchasing reacts to our purchase order (PO). If the buyers report to me directly, there is no doubt that they will attend to my PO. I will be the one who will evaluate the buyer’s performance. More importantly, his proximity to the job site will make him better understand the urgency and the nature of our requests. Communicating with him will be easier too. Sometimes, when the items I need are not available, he calls me to ask for substitutes. I always have problems reaching him by phone. If it’s not busy, he is not there! If my material are delayed, my project will not be completed as scheduled.† At this point during the workshop, the Purchasing Manager interrupted Mr. Remigio and said, â€Å"Our function is not merely to implement Pos. We purchase materials ordered by the projects only 1) if the items are not on stock in the central warehouse and 2) if the items are included in the Bill of Materials (BOM) as provided by the Executive Committee. Any order in excess of the approved by us unless there is an amended bill of materials. Therefore, if the buyers will report directly to project managers, this could lead to cost overruns. The President of the Company took the floor after hearing Mr. Remigio and the Purchasing Manager. â€Å"I am also concerned about the availability of funds for approved Pos to be acted upon by purchasing. While it is good for the projects to have the required materials on time, I wouldn’t want to encourage them to stock-up too much. This will tie-up funds and increase our interest costs. I hope that wherever Purchasing is to be located in the organization chart, we will also consider the matter of fund availability.† The Central warehouse is located in Antipolo while Head office (where the buyers gold office) is located in Paco, Manila. Approximately P10Million worth of materials are kept in the Central warehouse. Exhibit B PROJECT SITE ORGANIZATION CHART BOARD Equipment Engineering Superintendent Administration * General Foreman * Asst. Foreman Exhibit A GLOBE ORGANIZATION CHART * Marketing * Design * Estimating * Drafting * Accounting * Personnel * Finance * Treasury * Bookkeeper * Stock Clerk * Time Keeper Projects Construction Purchasing Project Development Administration Operations President BOARD

Immune Privilege of Tissue Engineered Articular Cartilage

Immune Privilege of Tissue Engineered Articular Cartilage The immune privilege of tissue engineered articular cartilage derived from mouse adult mesenchymal stem cells and the potential of tissue engineered cartilage as a gene delivery method Chapter 1 Stem cell biology 1.1 Categorization of stem cells Stem cells are generally defined as cells possessing the following 3 characteristics: (1) self-renewal, (2) the ability to produce all cell types made in that tissue, and (3) the ability to do so for a significant portion of the life of the host (Alberts et al., 1989; Reya et al., 2001), while progenitor cells are capable only of multi-lineage differentiation without self-renewal (Weissman, 2000). Stem cells can be classified by their ability to differentiate. The most primitive, totipotent stem cells have the ability to divide and produce all the differentiated cells in an organism, including both the embryonic and extraembryonic tissues of an organism. Totipotent stem cells include the fertilized egg and the cells produced by the initial divisions of it. In mammals, these cell divisions result in an implant in the uterus called the blastocyst. The blastocyst contains an outer sphere of trophoblast cells. Trophoblast cells are capable of implanting into the uterus and helping the form of placenta which provides nutrients to the embryo. Within the blastocyst are 10 to 20 pluripotent cells called the inner cell mass. In mammalian uterus, these inner mass cells will participate in the production of all tissues and organs of the developing embryo, then fetus, then born organism. Such pluripotent cells can produce any differentiated cells in the body, but are usually unable to for m the trophoblast cells. The best-known pluripotent stem cell is the embryonic stem (ES) cell, which are obtained from the inner cell mass of the blastocyst and exist for only a brief stage of embryonic development. The last major class of stem cells, multipotent stem cells, gives rise to a limited number of cell types which are responsible for organ growth and renewal such as neural stem cells, skin stem cells and haematopoietic stem cells (HSCs) (Cheshier et al., 2009). 1.2 Selected milestones of stem cell research In 1981, Martin isolated a pluripotent stem cell line from early mouse embryos (Martin, 1981). Wilmut in 1996 first cloned a mammal, a lamb named Dolly by transferring nuclear from the adult mammary gland cell to an enucleated unfertilized egg (Wilmut et al., 1997). In 1998, Thomson obtained the first human embryonic stem cell line from human blastocysts (Thomson et al., 1998). In 2001, President Bush banned scientists from using federal funds to study stem cells from sources other than those that had already been grown because of the ethical concerns. To avoid ethical dispute over the use of human embryonic cells for research purposes, many efforts have been taken on obtaining pluripotent stem cells from differentiated donor cells. In 2006, Yamanaka find a way to obtain pluripotent cells by reprogramming the nucleus of adult mice skin cells (Takahashi and Yamanaka, 2006). Such cells are now known as induced pluripotent stem (iPS) cells. 1.3 A brief introduction of several types of multipotent stem cell The best known multipotent stem cells are haematopoietic stem cells (HSCs), that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). HSCs are vital elements in bone-marrow transplantation, which has already been used extensively in therapeutic settings (Reya et al., 2001). In the long-term culture systems, human and rodent Central Neural System (CNS) cells maintain the capacity to produce the three main mature cell classes of the CNS: neurons, astrocytes, and oligodendrocytes, which suggest stem cells and/or progenitors exist and can survive in the culture medium (Weiss et al., 1996; Carpenter et al., 1999). In 2000, Human CNS stem cells (hCNS-SCs) have been successfully isolated by FACs (Uchida et al., 2000). Cancer stem cell hypothesis was proposed by Reya 2001 (Reya et al., 2001). This hypothesis consists of 2 components. The first component postulates that normal tissue stem cells are the target for transforming mutations and successive mutations result in the formation of a tumor. The second component is that within every cancer a specific subset of cancer stem cells continuously gives rise to all the other cancer cells and only these cells within a tumor possess the ability to self-renew, continuously proliferate. Conflicting to the first component of the hypothesis, evidences indicate cancer stem cells can also arise from mutated progenitor cells rather than stem cells (Cheshier et al., 2009). In addition, mature cells such as Lymphocytes can lead to mouse T cell leukemia independently from HSCs (Yuan et al., 2006). For the latter component of cancer stem cell hypothesis, it is likely that the cancer stem cell hypothesis is applicable to some tumors but not to others. In hematopoiet ic and some solid malignancies, only 1 in 100 to 1 in 10 000 primary tumor cells are capable of reproducing the tumor in vivo, such as human breast cancer, human neuroepithelial tumors, head and neck squamous cell carcinomas, and colon cancer. But in melanoma, nearly 1 in 4 cells possessed the ability of proliferation and developing into cancer (Cheshier et al., 2009). Cancer stem cells and CNS stem cells were reviewed by Cheshier et al. (Cheshier et al., 2009). 1.4 Mesenchymal stem cells (MSCs) and their differentiation potential Bone marrow is composed of two main systems of cell, hematopoietic cells and the supporting stromal cells (Bianco et al., 2001). MSCs reside within the marrow, maintain a level of self-renewal, and give rise to progenitor cells that can differentiate into various lineages of tissue, including chondrocytes, osteoblasts, adipocytes, fibroblasts, marrow stroma, and other tissues of mesenchymal origin. The traditional opinion about the multipotent differentiation potential of MSCs was challenged by further studies. Interestingly, MSCs reside in a diverse host of tissues throughout the adult organism and possess the ability to ‘regenerate cell types specific for local tissues e.g. adipose, periosteum, synovial membrane, muscle, dermis, pericytes, blood, bone marrow, and most recently trabecular bone, reviewed by Tuan et al. (Tuan et al., 2003). Furthermore, in 2002, Jiang et al. reported a rare cell within human bone marrow mesenchymal stem cell cultures that can be expanded extensi vely without obvious senescence. This cell population can differentiate, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. Most somatic cell types could be derived after this population of cells was injected into an early blastocyst (Jiang et al., 2002). These studies suggest mesenchymal stem cells maintained pluripotent properties. Chapter 2 Features of Articular Cartilage 2.1 Introduction Joint cartilage formed highly sophisticated structure during the evolutionary development. There have been considerable research interests related to the cartilage cells, chondrocytes. In the last decades these studies made cartilage the first and very successful tissue engineering treatment (Brittberg et al. 1994). 2.2 Categorization of cartilage tissues Cartilage tissue is categorised in three major types by different biochemical compositions and structures of their extracellular matrix (ECM). Elastic cartilage has a small concentration of proteoglycans (PGs), and a relatively high proportion of elastin fibres. It exists in the epiglottis, small laryngeal, the external ear, auditory tube, and the small bronchi, where it is generally required to resist bending forces. Fibrocartilage also possesses a small concentration of PGs, but far less elastins. The meniscus in the knee joint is made of fibrocartilage. Hyaline is the most widespread cartilage in the human body. It is resistant to compressive or tensile forces due to its special type II collagen fibril mesh filled with a high concentration of PGs. Hyaline cartilage can be found in the nose, trachea, bronchi, and synovial joints. In the latter case, it is termed as articular cartilage (Schulz and Bader, 2007). 2.3 Compositions of articular cartilage Chondrocytes contribute to only 1%- 5% of the tissue volume; the remaining 95%-99% being extracellular matrix (ECM). Chondrocytes sense and synthesize all necessary ECM components (Mollenhauer, 2008; Schulz and Bader, 2007). The ECM of articular cartilage consists of about 60-85% water and dissolved electrolytes. The solid framework is composed of collagens (10-20%), PGs (3-10%), noncollagenous proteins and glycoproteins. In articular cartilage, 95% of collagen in the ECM is type II collagen fibrils. The rest other types are collagen type IX and XI and a small fraction of types III, VI, XII and XIV. Normal articular cartilage does not present type I collagen, which is concerned with fibrous tissue. Unlike Type I and Type III collagens which form thick fibres and thin  ¬Ã‚ bres respectively, Type II collagen present in hyaline and elastic cartilages does not form  ¬Ã‚ bres. It forms very thin  ¬Ã‚ brils which are disposed as a loose mesh that strongly interacts with the groun d substance. Type II collagen provides tensile stiffness and strength to articular cartilage and constrains the swelling capacity generated by highly negatively charged glycosaminoglycans (GAGs) of the proteoglycans (PGs). The majority (50-85%) of the PG content in articular cartilage were presented by large molecule aggrecan. It consists of a protein backbone, the core protein, to which unbranched GAGs side chains of chondroitin sulphate (CS) and keratan sulfate (KS) are covalently attached (Figure 1.1). The composition of articular cartilage was extensively reviewed by Schulz and Bader (Schulz and Bader, 2007). Figure 1. Illustration of the extracellular matrix (ECM) organization of articular cartilage (Left) and the schematic sketches (Right) of the most relevant polysaccharides of proteoglycans (PGs) in articular cartilage. The PGs consist of a strand of hyaluronic acid (HA), to which a core protein is non-covalently attached. On the core protein, glycosaminoglycans (GAGs) such as keratan sulphate (KS) and chondroitin sulfate (CS) are covalently bound in a bottle brush fashion (Modified from Schulz and Bader, 2007 and Mow and Wang, 1999). 2.4 Low capacity of self-repair in articular cartilage The aneural and avascular nature of articular cartilage, coupled with its low cellularity, contribute to both the limited rate and incomplete nature of the repair process following damage (Heywood et al., 2004). The low mitotic potential of chondrocytes in vivo also contributes to its poor ability to undergo self-repair (Kuroda et al., 2007). Some researchers believe that cartilage lesions less than 3mm in diameter self-repair with normal hyaline-like cartilage (Revell and Athanasiou, 2009; Schulz and Bader, 2007). In animal studies, full thickness cartilage defects, extending into the subchondral bone, have been reported to heal with the formation of fibrous tissue, which contains relatively low amount of type II collagen and aggrecan, but a relatively high concentration of type I collagen which is not present in normal adult articular cartilage and accordingly exhibits impaired mechanical properties (Hjertquist et al., 1971). 2.5 Metabolism of articular cartilage Joint cartilage is supplied with nutrients and oxygen by the synovial fluid diffusion facilitated by compressive cyclic loading during joint movements which acts as a pumping function (Mollenhauer, 2008). Within synovial joints, oxygen supply to articular chondrocytes is very limited, from 7.5% at the superficial zone down to 1% oxygen tension at the deep zone. It is supposed to be even further decreased under pathological conditions, such as osteoarthritis (OA) or rheumatoid arthritis (RA). The metabolism of chondrocytes is largely glycolytic. Oxygen-dependent energy generated by oxidative phosphorylation is just a minor contributor to the overall energy in chondrocytes. Nevertheless, changes in O2 tension have profound effects on cell metabolism, phenotype, gene expression, and morphology, as well as response to, and production of, cytokines (Pfander and Gelse, 2007; Gibson et al., 2008). The most important component of this hypoxic response is mediated by transcription factor hypo xia-inducible factor-1 (HIF-1), which is present in most hypoxia inducible genes (Pfander and Gelse, 2007; Gibson et al., 2008). Moreover, the matrix turnover in articular cartilage is extremely slow. Proteoglycan turnover is up to 25 years. Collagen half-life is estimated to range from several decades up to 400 years (Mollenhauer, 2008). Chapter 3 Osteoarthritis (OA) 3.1 Prevalence Osteoarthritis (OA) is the most common form of arthritis. More than 40 million US American citizens (approximately 15% of the overall population of the USA) suffer from arthritis (Schulz and Bader, 2007). OA can occur in any joint but is most common in certain joints of the hand, knee, foot and hip. OA is the most common reason for total hip- and knee-joint replacement (Wieland et al., 2005). Among US adults 30 years of age or older, symptomatic disease in the knee occurs in approximately 6% and symptomatic hip osteoarthritis in roughly 3% (Felson and Zhang, 1998). 3.2 The symptoms and diagnosis The symptoms of OA include pain, stiffness and loss of function. OA can be monitored by radiography, magnetic resonance imaging (MRI), and arthroscopy, but radiographs are still considered the gold standard (Wieland et al., 2005). 3.3 The pathology of OA The pathologic characteristics of OA are the slowly developing degenerative breakdown of cartilage; the pathological changes in the bone, including osteophyte formation and thickening of the subchondral plate; the changes in the synovium such as inflammatory infiltrates; ligaments, which are often lax; and bridging muscle, which becomes weak. Many people with pathologic and radiographic evidence of osteoarthritis have no symptoms (Martel-Pelletier, 1999; Felson et al., 2000). A protease family of matrix metalloproteases (MMP) is responsible for the initial occurrence of cartilage matrix digestion. Of this family, collagenases, the stromelysins and the gelatinases are identified as being elevated in OA. Another group of MMP is localized at the cell membrane surface and is thus named membrane type MMP (MT-MMP) (Martel-Pelletier, 1999). Proinflamatory cytokines such as interleukin (IL)-1ÃŽ ², Tumor necrosis factor (TNF)-ÃŽ ±, IL-6, leukemic inhibitor factor (LIF) and IL-17 are first produced by the synovial membrane and then diffuse into the cartilage through the synovial fluid, where they activate the chondrocytes to produce proinflammatory cytokines. These proinflamatory cytokines are considered responsible for the catabolic pathological process (Martel-Pelletier, 1999). In OA cartilage, an increased level of an inducible form of nitric oxide synthase (iNOS) leads to a large amount of nitric oxide (NO) production (Pelletier et al., 2001). NO can inhibit the synthesis of cartilage matrix macromolecules such as aggrecans and can enhance MMP activity (Taskiran et al., 1994; Murrell et al., 1995). It is well stablished that proinflammatory cytokines such as IL-1ÃŽ ² act as the key mediators of cartilage breakdown and stimulate the release of inflammatory products (NO) and prostaglandin (PG)E2, via induction of iNOS and cyclo-oxygenase (COX)-2 enzymes (Chowdhury et al., 2008). 3.4 Risk factors Osteoarthritis is considered to be a systemic disease although severe joint injury may be sufficient to cause osteoarthritis. There are several systemic risk factors related to OA. (1) Age: Osteoarthritis increases with ages, the incidence and prevalence of disease increased 2- to 10-fold from 30 to 65 years of age and increased further thereafter in a community-based survey (Oliveria et al., 1995). (2) Hormonal status and bone density: women taking estrogen have a decreased prevalence of radiographic osteoarthritis (Nevitt et al., 1996). Before 50 years of age, the prevalence of osteoarthritis in most joints is higher in men than in women. After about age 50 years, women are more often affected with hand, foot, and knee osteoarthritis than men. In most studies, hip osteoarthritis is more frequent in men (van Saase et al., 1989). Evidence suggests an inverse relationship between osteoarthritis and osteoporosis (Felson et al., 2000). (3) Nutritional factors: evidence indicates that co ntinuous exposure to oxidants contributes to the development of many common age-related diseases, including osteoarthritis. McAlindon et al. reported a threefold reduction in risk for progressive radiographic osteoarthritis was observed in persons in the middle and highest tertile of vitamin C intake compared with those whose intake was in the lowest tertile (McAlindon et al., 1996a). Vitamin D intake was observed associated with the progression of OA although not associated with risk for new-onset radiographic osteoarthritis (McAlindon et al., 1996b; Lane et al., 1999). (4) Genetics: genetic factors account for at least 50% of cases of osteoarthritis in the hands and hips and a smaller percentage in the knees (Spector et al., 1996). Candidate genes for common forms of osteoarthritis include the vitamin D receptor gene, insulin-like growth factor I genes, cartilage oligomeric protein genes, and the HLA region (Felson et al., 2000). Local mechanical factors include the body weight and the pathological alterations of the mechanical environment of the joint. Persons who are overweight have a high prevalence of knee osteoarthritis (Felson et al., 1997). OA is also considered to be related to alterations in joint mechanical environments such as knee laxity, the displacement or rotation of the tibia with respect to the femur; proprioception, the conscious and unconscious perception of joint position and movement; knee alignment , knee position in reference to the hip and ankle (Felson et al., 2000). In addition, joint dysplasias, fractures of articular surfaces, and tears of menisci and ligaments that increase joint instability precede the development of osteoarthritis in a high percentage of affected joints. Risk factors for posttraumatic osteoarthritis include high body mass, high level of activity, residual joint instability or malalignment, and persistent articular surface incongruity (Buckwalter et al., 1997; Honkonen 1995). 3.5 Treatments The medicine treatment of OA was dominated by COX2 inhibitors (Flower 2003). The other medicines include glucosamine, chondroitin (McAlindon et al., 2000), and hyaluronic acid (Lo et al., 2003). In addition, both aerobic walking and muscle strengthening exercise reduce pain and disability from osteoarthritis (Roddy et al., 2005). Articular cartilage lesions, both of traumatic or pathological origin, do not heal spontaneously and often undergo progressive degeneration towards osteoarthritis (OA). The most frequently used treatments include the artificial joint replacement, mosaicplasty, marrow stimulation, and autologous condrocyte implantation (ACI) (Steinwachs et al., 2008). Total joint replacement is most commonly performed in people over 60 years of age. (NHS 2006; Brittberg et al., 1994) Mosaicplasty is an autologous osteochondral transplantation method through which cylindrical periosteum grafts are taken from periphery of the patellofemoral area which bears less weight, and transplanted to defective areas. This transplantation can be done with various diameters of grafts (Haklar et al., 2008; NHS, 2006). Marrow stimulation methods include arthroscopic surgery to smooth the surface of the damaged cartilage area; microfracture, drilling, abrasion. All marrow stimulation methods base on the penetration of the subchondral bone plate at the bottom of the cartilage defect. The outflowing bone marrow blood contains the mesenchymal stem cells which are stabilised by the clot formation in the defect. These pluripotent stem cells which are able to differentiate into fibrochondrocytes, result in fibrocartilage repair with varying amounts of type I, II and III collagen (Steinwachs et al., 2008). The ACI tissue engineering treatment will be discussed in the next chapter. Chapter 4 Tissue engineering and autologous chondrocyte implantation (ACI) 4.1 Overview of tissue engineering technologies Tissue engineering is defined as ‘‘the application of the principles and methods of engineering and the life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve tissue function† (Langer and Vacanti, 1993). Three factors are considered as the principles of tissue engineering, including the utilization of biocompatible and mechanically suitable scaffolds, an appropriate cell source, and bioactive molecules to promote the differentiation and maturation of the cell type of interest (Song et al., 2004). Potential applications of tissue engineering are involved in the following fields: skin, cartilage, bone, cardiovascular diseases, organs (e.g. liver, pancreas, bladder, trachea and breast), central nervous system (e.g. spinal cord), and miscellaneous (e.g. soft tissue, ligaments). Although research is being carried out in all these fields, only few products have already entered the market. The most successful products up to now are: tissue engineered skin which is mainly used for wound cover, autologous chondrocyte implantation (ACI), and artificial bone graft (Hà ¼sing et al., 2003). 4.2 Autologous chondrocyte implantation (ACI) In 1984, a study in rabbits reported successful treatment of focal patellar defects with the use of ACI. One year after transplantation, newly formed cartilage-like tissue typically covered about 70 percent of the defect (Grande et al. 1989). In 1987, Brittberg firstly performed ACI in 23 people with deep cartilage defects in the knee. ACI is described as the following procedure: cartilage cells are taken from a minor load-bearing area on the upper medial femoral condyle of the damaged knee via an arthroscopic procedure, cultivated for four to six weeks in a laboratory and then, in open surgery, introduced back into the damaged area as a liquid or mesh-like transplant; at last, a periosteal flap sutured in place to secure the transplant (Figure 2; Brittberg et al., 1994). Genzyme Biosurgery with its product Carticel ® was the first company which introduced ACI into market and is the market leader in USA. Carticel ® is a classic ACI procedure using the periosteal cover (Hà ¼sing et al., 2008). Today the periosteum is often replaced by an artificial resorbable cover such as collagen I/III and hyaluronan membrane, such as ChondroGide or Restore (De Puy, Warzaw, Indiana) (Gooding et al., 2006; Jones and Peterson, 2006). Another new method uses chondrocytes cultured on a tri-dimensional (3D), biodegradable scaffold. This kind of scaffold, cut to the required size, is fixed into the lesion by anchoring stitches or its sticky nature. The 3D cell seeded scaffold eliminates the using of cover, thus simplifies the surgery procedure, saves the surgery time, and opens up the possibility of an arthroscopic surgery instead of the open surgery which causes more tissue damage. HYALOGRAFT from Italy is one of the European market leaders. It is a cartilage substit ute made of autologous chondrocytes delivered on a biocompatible 3D matrix, entirely composed of a derivative of hyaluronic acid (Marcacci et al. 2005). 4.3 Clinical results of ACI Brittberg studied the long-term durability of ACI-treated patients, 61 patients were followed for at least five years up to 11 years post-surgery (mean 7.4 years). After two years, 50 out of 61 patients were graded good-excellent. At the five to 11 years follow-up, 51 of the 61 were graded good-excellent (Brittberg et al., 2003). Since 1997 the year FDA approved ACI, this method has been widely performed in more than 20,000 patients all over the world. It has been reported to be effective in relieving clinical symptoms, such as pain and function (Wakitani et al., 2008). In a randomised controlled study, Knutsen et al. studied 80 patients who needed local cartilage repair with lesions on the femoral condyles of 2-10 cm2. There were no signi ¬Ã‚ cant differences in clinical results at 5 years follow-up (Knutsen et al., 2007). In another randomised controlled study that compared mosaicplasty with ACI, there was no significant difference in the number of patients who had an excellent or good clinical outcome at 1 year (69% [29/42] and 88% [51/58], respectively). In the subgroup of patients who had repairs to lesions of the medial femoral condyle, significantly more patients who had ACI had an excellent or good outcome (88% [21/24]) compared with those who had mosaicplasty (72% [21/29]) (p Clinical results of ACI were reviewed by Gikas 2009 (Gikas et al., 2009). Generally speaking, the outcomes of ACI treatment have been encouraging. However, most randomised controlled studies showed no significant difference between ACI and traditional treatments. 4.4 Limitations of ACI Microfracture is a very simple and low-cost procedure whereas ACI costs about $10 000 per patient. If ACI is not found to be more effective for improving articular cartilage repair than microfracture, the procedure will not be continued (Wakitani et al., 2008). There are several possible reasons to be blamed for the limitations of the traditional ACI procedure. The cell source in ACI is the cartilage tissue derived via an arthroscopic procedure from the low load-bearing area on the upper medial femoral condyle of the damaged knee. However, Wiseman et al. found the chondrocytes isolated from the low loaded area of the knee joint respond to mechanical stimulations in a distinct manner with the chondrocytes from the high loaded area, which suggests the traditional cell source of ACI may not provide enough mechanical response and may further lead to the insufficient mechanical properties of the repaired tissue (Wiseman et al. 2003). As cultured in monolayer, chondrocytes undergo a process of dedifferentiation and adopt a more  ¬Ã‚ broblast-like morphology, which is accompanied by an increase in proliferation and an altered phenotype. Type II collagen, the major protein produced by chondrocytes in articular cartilage, are down-regulated in the culture, while collagen types I and III are increased (Glowacki et al., 1983; Stocks et al., 2002; Benya et al., 1978). The agregating proteoglycan aggrecan of articular cartilage, is down-regulated during dedifferentiation and replaced by proteoglycans not speci ¬Ã‚ c to cartilage, such as versican (Glowacki et al., 1983; Stocks et al., 2002). Therefore, monolayer cultured chondrocytes do not express the origninal phenotype, and their ability to regenerate damaged cartilage tissue is impaired. Upon implantation, dedifferentiated cells may form a  ¬Ã‚ brous tissue expressing collagen type I that does not have appropriate mechanical properties, which may lead to deg radation and failure of the repair tissue (Brodkin et al., 2004). Chondrocytes grown in conditions that support their round shape, such as plating in high-density monolayer (Watt, 1988) and seeding in 3D structure (Benya and Shaffer, 1982) can maintain their differentiated phenotype much longer compared to cells spread in monolayer cultures. Although ACI can still be considered to be one of commonly form of repair of cartilage defects, it does have a number of scientific limitations. Some of those can be resolved using more comprehensive tissue engineered strategies which incorporates cells, scaffold materials and potentially biochemical, biomechanical and/or physical stimulation in a controlled bioreactor environment. 4.5 Tissue engineering strategies for ACI Chondrocytes derived from the low load bearing area of the knee joint respond in a distinct manner with the chondrocytes from the high loaded area. Chondrocytes cultured in monolayer have a dedifferentiation phenomenon as described above. In addition, the limitation of the transplant volume is always a major problem in autograft to be overcome (Kitaoka et al., 2001; Vinatier et. al, 2009). Accordingly, potential cell sources are widely studied for the future improvement of ACI approach, which will be discussed in Chapter 4. Seeding in 3D structures (Benya and Shaffer, 1982) can maintain chondrocytes differentiated phenotype. Ideally, cell scaffolds for tissue engineering should meet several design criteria: (1) The surface should permit cell adhension and growth, (2) neither the polymer nor its degradation products should provoke inflammation or toxicity when implanted in vivo, (3) the material should be reproducibly processable into three dimensional structures, (4) the porosity should be at least 90% in order to provide a high surface area for cell-polymer interactions, sufficient space of extracellular matrix regeneration, and minimal diffusional constraints during in vitro culture, (5) the scaffold should resorb once it has served its purpose of providing a template for the regenerating tissue, since foreign materials carry a permanent risk of inflammation, and (6) the scaffold degradation rate should be adjustable to match the rate of tissue regeneration by the cell type of interest (Freed et al., 1994). Synthetic materials such as poly (glycolic acid) (PGA), poly (lactic acid) (PLA), and poly (lactic-co-glycolic acid) (PLGA) have been investigated for use as cartilage tissue engineering scaffolds (Cima et al., 1991; Vacanti et al., 1991). Both, in vitro and in vivo studies have demonstrated these scaffold maintained the chondrocyte phenotype and the production of cartilage extracellular matrix (ECM) (Barnewitz et al., 2006; Kaps et al., 2006). Moreover, PLGA is used as a scaffold material for matrix-based autologous chondrocyte transplantation clinically (Ossendorf et al., 2007). Natural materials have also been investigated in the application of tissue engineering scaffolds in ACI. Collagen-based biomaterials are widely used in todays clinical practice (for example, haemostasis and cosmetic surgery). Collagen is also be commonly used as main components in tissue engineered skin products. Several commercial ACI products have used collagenous membraneas as the replacement for the periosteum to close the defect, such as ChondroGide or Restore (De Puy, Warzaw, Indiana) (Cicuttini et al., 1996; Jones and Peterson, 2006). The combination of type I collagen with GAG in scaffolds had a positive effect on chondrocyte phenotype (van Susante et al., 2001). Hyaluronic acid is a non-sulphated GAG that makes up a large proportion of cartilage extracellular matrix (Schulz and Bader, 2007). Matrices composed of hyaluronan have been frequently used as carriers for chondrocytes. Facchini et al. con ¬Ã‚ rms the hyaluronan derivative scaffold Hyaff  ®11 as a suitable scaff old both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects in their study (Facchini et al., 2006). Sugar-based natural polymers such as chitosan, alginate and agarose can be formulated as hydrogels and in some cases sponges or pads. Although these materials are extensively used in in vitro research, their role in in vivo cartilage reconstruction is still limited (Stoop, 2008). Growth factors are proved to be able to promote the formation of new cartilage tissue in both explants and engineered constructs. Insulin-like growth factor-I (IGF-I), transforming growth factor-ÃŽ ²1 (TGF-ÃŽ ²1) increases, basic fibroblast growth factor (bFGF) can stimulate cell proliferation and/or biosynthesis in chondrocytes which were Immune Privilege of Tissue Engineered Articular Cartilage Immune Privilege of Tissue Engineered Articular Cartilage The immune privilege of tissue engineered articular cartilage derived from mouse adult mesenchymal stem cells and the potential of tissue engineered cartilage as a gene delivery method Chapter 1 Stem cell biology 1.1 Categorization of stem cells Stem cells are generally defined as cells possessing the following 3 characteristics: (1) self-renewal, (2) the ability to produce all cell types made in that tissue, and (3) the ability to do so for a significant portion of the life of the host (Alberts et al., 1989; Reya et al., 2001), while progenitor cells are capable only of multi-lineage differentiation without self-renewal (Weissman, 2000). Stem cells can be classified by their ability to differentiate. The most primitive, totipotent stem cells have the ability to divide and produce all the differentiated cells in an organism, including both the embryonic and extraembryonic tissues of an organism. Totipotent stem cells include the fertilized egg and the cells produced by the initial divisions of it. In mammals, these cell divisions result in an implant in the uterus called the blastocyst. The blastocyst contains an outer sphere of trophoblast cells. Trophoblast cells are capable of implanting into the uterus and helping the form of placenta which provides nutrients to the embryo. Within the blastocyst are 10 to 20 pluripotent cells called the inner cell mass. In mammalian uterus, these inner mass cells will participate in the production of all tissues and organs of the developing embryo, then fetus, then born organism. Such pluripotent cells can produce any differentiated cells in the body, but are usually unable to for m the trophoblast cells. The best-known pluripotent stem cell is the embryonic stem (ES) cell, which are obtained from the inner cell mass of the blastocyst and exist for only a brief stage of embryonic development. The last major class of stem cells, multipotent stem cells, gives rise to a limited number of cell types which are responsible for organ growth and renewal such as neural stem cells, skin stem cells and haematopoietic stem cells (HSCs) (Cheshier et al., 2009). 1.2 Selected milestones of stem cell research In 1981, Martin isolated a pluripotent stem cell line from early mouse embryos (Martin, 1981). Wilmut in 1996 first cloned a mammal, a lamb named Dolly by transferring nuclear from the adult mammary gland cell to an enucleated unfertilized egg (Wilmut et al., 1997). In 1998, Thomson obtained the first human embryonic stem cell line from human blastocysts (Thomson et al., 1998). In 2001, President Bush banned scientists from using federal funds to study stem cells from sources other than those that had already been grown because of the ethical concerns. To avoid ethical dispute over the use of human embryonic cells for research purposes, many efforts have been taken on obtaining pluripotent stem cells from differentiated donor cells. In 2006, Yamanaka find a way to obtain pluripotent cells by reprogramming the nucleus of adult mice skin cells (Takahashi and Yamanaka, 2006). Such cells are now known as induced pluripotent stem (iPS) cells. 1.3 A brief introduction of several types of multipotent stem cell The best known multipotent stem cells are haematopoietic stem cells (HSCs), that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). HSCs are vital elements in bone-marrow transplantation, which has already been used extensively in therapeutic settings (Reya et al., 2001). In the long-term culture systems, human and rodent Central Neural System (CNS) cells maintain the capacity to produce the three main mature cell classes of the CNS: neurons, astrocytes, and oligodendrocytes, which suggest stem cells and/or progenitors exist and can survive in the culture medium (Weiss et al., 1996; Carpenter et al., 1999). In 2000, Human CNS stem cells (hCNS-SCs) have been successfully isolated by FACs (Uchida et al., 2000). Cancer stem cell hypothesis was proposed by Reya 2001 (Reya et al., 2001). This hypothesis consists of 2 components. The first component postulates that normal tissue stem cells are the target for transforming mutations and successive mutations result in the formation of a tumor. The second component is that within every cancer a specific subset of cancer stem cells continuously gives rise to all the other cancer cells and only these cells within a tumor possess the ability to self-renew, continuously proliferate. Conflicting to the first component of the hypothesis, evidences indicate cancer stem cells can also arise from mutated progenitor cells rather than stem cells (Cheshier et al., 2009). In addition, mature cells such as Lymphocytes can lead to mouse T cell leukemia independently from HSCs (Yuan et al., 2006). For the latter component of cancer stem cell hypothesis, it is likely that the cancer stem cell hypothesis is applicable to some tumors but not to others. In hematopoiet ic and some solid malignancies, only 1 in 100 to 1 in 10 000 primary tumor cells are capable of reproducing the tumor in vivo, such as human breast cancer, human neuroepithelial tumors, head and neck squamous cell carcinomas, and colon cancer. But in melanoma, nearly 1 in 4 cells possessed the ability of proliferation and developing into cancer (Cheshier et al., 2009). Cancer stem cells and CNS stem cells were reviewed by Cheshier et al. (Cheshier et al., 2009). 1.4 Mesenchymal stem cells (MSCs) and their differentiation potential Bone marrow is composed of two main systems of cell, hematopoietic cells and the supporting stromal cells (Bianco et al., 2001). MSCs reside within the marrow, maintain a level of self-renewal, and give rise to progenitor cells that can differentiate into various lineages of tissue, including chondrocytes, osteoblasts, adipocytes, fibroblasts, marrow stroma, and other tissues of mesenchymal origin. The traditional opinion about the multipotent differentiation potential of MSCs was challenged by further studies. Interestingly, MSCs reside in a diverse host of tissues throughout the adult organism and possess the ability to ‘regenerate cell types specific for local tissues e.g. adipose, periosteum, synovial membrane, muscle, dermis, pericytes, blood, bone marrow, and most recently trabecular bone, reviewed by Tuan et al. (Tuan et al., 2003). Furthermore, in 2002, Jiang et al. reported a rare cell within human bone marrow mesenchymal stem cell cultures that can be expanded extensi vely without obvious senescence. This cell population can differentiate, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. Most somatic cell types could be derived after this population of cells was injected into an early blastocyst (Jiang et al., 2002). These studies suggest mesenchymal stem cells maintained pluripotent properties. Chapter 2 Features of Articular Cartilage 2.1 Introduction Joint cartilage formed highly sophisticated structure during the evolutionary development. There have been considerable research interests related to the cartilage cells, chondrocytes. In the last decades these studies made cartilage the first and very successful tissue engineering treatment (Brittberg et al. 1994). 2.2 Categorization of cartilage tissues Cartilage tissue is categorised in three major types by different biochemical compositions and structures of their extracellular matrix (ECM). Elastic cartilage has a small concentration of proteoglycans (PGs), and a relatively high proportion of elastin fibres. It exists in the epiglottis, small laryngeal, the external ear, auditory tube, and the small bronchi, where it is generally required to resist bending forces. Fibrocartilage also possesses a small concentration of PGs, but far less elastins. The meniscus in the knee joint is made of fibrocartilage. Hyaline is the most widespread cartilage in the human body. It is resistant to compressive or tensile forces due to its special type II collagen fibril mesh filled with a high concentration of PGs. Hyaline cartilage can be found in the nose, trachea, bronchi, and synovial joints. In the latter case, it is termed as articular cartilage (Schulz and Bader, 2007). 2.3 Compositions of articular cartilage Chondrocytes contribute to only 1%- 5% of the tissue volume; the remaining 95%-99% being extracellular matrix (ECM). Chondrocytes sense and synthesize all necessary ECM components (Mollenhauer, 2008; Schulz and Bader, 2007). The ECM of articular cartilage consists of about 60-85% water and dissolved electrolytes. The solid framework is composed of collagens (10-20%), PGs (3-10%), noncollagenous proteins and glycoproteins. In articular cartilage, 95% of collagen in the ECM is type II collagen fibrils. The rest other types are collagen type IX and XI and a small fraction of types III, VI, XII and XIV. Normal articular cartilage does not present type I collagen, which is concerned with fibrous tissue. Unlike Type I and Type III collagens which form thick fibres and thin  ¬Ã‚ bres respectively, Type II collagen present in hyaline and elastic cartilages does not form  ¬Ã‚ bres. It forms very thin  ¬Ã‚ brils which are disposed as a loose mesh that strongly interacts with the groun d substance. Type II collagen provides tensile stiffness and strength to articular cartilage and constrains the swelling capacity generated by highly negatively charged glycosaminoglycans (GAGs) of the proteoglycans (PGs). The majority (50-85%) of the PG content in articular cartilage were presented by large molecule aggrecan. It consists of a protein backbone, the core protein, to which unbranched GAGs side chains of chondroitin sulphate (CS) and keratan sulfate (KS) are covalently attached (Figure 1.1). The composition of articular cartilage was extensively reviewed by Schulz and Bader (Schulz and Bader, 2007). Figure 1. Illustration of the extracellular matrix (ECM) organization of articular cartilage (Left) and the schematic sketches (Right) of the most relevant polysaccharides of proteoglycans (PGs) in articular cartilage. The PGs consist of a strand of hyaluronic acid (HA), to which a core protein is non-covalently attached. On the core protein, glycosaminoglycans (GAGs) such as keratan sulphate (KS) and chondroitin sulfate (CS) are covalently bound in a bottle brush fashion (Modified from Schulz and Bader, 2007 and Mow and Wang, 1999). 2.4 Low capacity of self-repair in articular cartilage The aneural and avascular nature of articular cartilage, coupled with its low cellularity, contribute to both the limited rate and incomplete nature of the repair process following damage (Heywood et al., 2004). The low mitotic potential of chondrocytes in vivo also contributes to its poor ability to undergo self-repair (Kuroda et al., 2007). Some researchers believe that cartilage lesions less than 3mm in diameter self-repair with normal hyaline-like cartilage (Revell and Athanasiou, 2009; Schulz and Bader, 2007). In animal studies, full thickness cartilage defects, extending into the subchondral bone, have been reported to heal with the formation of fibrous tissue, which contains relatively low amount of type II collagen and aggrecan, but a relatively high concentration of type I collagen which is not present in normal adult articular cartilage and accordingly exhibits impaired mechanical properties (Hjertquist et al., 1971). 2.5 Metabolism of articular cartilage Joint cartilage is supplied with nutrients and oxygen by the synovial fluid diffusion facilitated by compressive cyclic loading during joint movements which acts as a pumping function (Mollenhauer, 2008). Within synovial joints, oxygen supply to articular chondrocytes is very limited, from 7.5% at the superficial zone down to 1% oxygen tension at the deep zone. It is supposed to be even further decreased under pathological conditions, such as osteoarthritis (OA) or rheumatoid arthritis (RA). The metabolism of chondrocytes is largely glycolytic. Oxygen-dependent energy generated by oxidative phosphorylation is just a minor contributor to the overall energy in chondrocytes. Nevertheless, changes in O2 tension have profound effects on cell metabolism, phenotype, gene expression, and morphology, as well as response to, and production of, cytokines (Pfander and Gelse, 2007; Gibson et al., 2008). The most important component of this hypoxic response is mediated by transcription factor hypo xia-inducible factor-1 (HIF-1), which is present in most hypoxia inducible genes (Pfander and Gelse, 2007; Gibson et al., 2008). Moreover, the matrix turnover in articular cartilage is extremely slow. Proteoglycan turnover is up to 25 years. Collagen half-life is estimated to range from several decades up to 400 years (Mollenhauer, 2008). Chapter 3 Osteoarthritis (OA) 3.1 Prevalence Osteoarthritis (OA) is the most common form of arthritis. More than 40 million US American citizens (approximately 15% of the overall population of the USA) suffer from arthritis (Schulz and Bader, 2007). OA can occur in any joint but is most common in certain joints of the hand, knee, foot and hip. OA is the most common reason for total hip- and knee-joint replacement (Wieland et al., 2005). Among US adults 30 years of age or older, symptomatic disease in the knee occurs in approximately 6% and symptomatic hip osteoarthritis in roughly 3% (Felson and Zhang, 1998). 3.2 The symptoms and diagnosis The symptoms of OA include pain, stiffness and loss of function. OA can be monitored by radiography, magnetic resonance imaging (MRI), and arthroscopy, but radiographs are still considered the gold standard (Wieland et al., 2005). 3.3 The pathology of OA The pathologic characteristics of OA are the slowly developing degenerative breakdown of cartilage; the pathological changes in the bone, including osteophyte formation and thickening of the subchondral plate; the changes in the synovium such as inflammatory infiltrates; ligaments, which are often lax; and bridging muscle, which becomes weak. Many people with pathologic and radiographic evidence of osteoarthritis have no symptoms (Martel-Pelletier, 1999; Felson et al., 2000). A protease family of matrix metalloproteases (MMP) is responsible for the initial occurrence of cartilage matrix digestion. Of this family, collagenases, the stromelysins and the gelatinases are identified as being elevated in OA. Another group of MMP is localized at the cell membrane surface and is thus named membrane type MMP (MT-MMP) (Martel-Pelletier, 1999). Proinflamatory cytokines such as interleukin (IL)-1ÃŽ ², Tumor necrosis factor (TNF)-ÃŽ ±, IL-6, leukemic inhibitor factor (LIF) and IL-17 are first produced by the synovial membrane and then diffuse into the cartilage through the synovial fluid, where they activate the chondrocytes to produce proinflammatory cytokines. These proinflamatory cytokines are considered responsible for the catabolic pathological process (Martel-Pelletier, 1999). In OA cartilage, an increased level of an inducible form of nitric oxide synthase (iNOS) leads to a large amount of nitric oxide (NO) production (Pelletier et al., 2001). NO can inhibit the synthesis of cartilage matrix macromolecules such as aggrecans and can enhance MMP activity (Taskiran et al., 1994; Murrell et al., 1995). It is well stablished that proinflammatory cytokines such as IL-1ÃŽ ² act as the key mediators of cartilage breakdown and stimulate the release of inflammatory products (NO) and prostaglandin (PG)E2, via induction of iNOS and cyclo-oxygenase (COX)-2 enzymes (Chowdhury et al., 2008). 3.4 Risk factors Osteoarthritis is considered to be a systemic disease although severe joint injury may be sufficient to cause osteoarthritis. There are several systemic risk factors related to OA. (1) Age: Osteoarthritis increases with ages, the incidence and prevalence of disease increased 2- to 10-fold from 30 to 65 years of age and increased further thereafter in a community-based survey (Oliveria et al., 1995). (2) Hormonal status and bone density: women taking estrogen have a decreased prevalence of radiographic osteoarthritis (Nevitt et al., 1996). Before 50 years of age, the prevalence of osteoarthritis in most joints is higher in men than in women. After about age 50 years, women are more often affected with hand, foot, and knee osteoarthritis than men. In most studies, hip osteoarthritis is more frequent in men (van Saase et al., 1989). Evidence suggests an inverse relationship between osteoarthritis and osteoporosis (Felson et al., 2000). (3) Nutritional factors: evidence indicates that co ntinuous exposure to oxidants contributes to the development of many common age-related diseases, including osteoarthritis. McAlindon et al. reported a threefold reduction in risk for progressive radiographic osteoarthritis was observed in persons in the middle and highest tertile of vitamin C intake compared with those whose intake was in the lowest tertile (McAlindon et al., 1996a). Vitamin D intake was observed associated with the progression of OA although not associated with risk for new-onset radiographic osteoarthritis (McAlindon et al., 1996b; Lane et al., 1999). (4) Genetics: genetic factors account for at least 50% of cases of osteoarthritis in the hands and hips and a smaller percentage in the knees (Spector et al., 1996). Candidate genes for common forms of osteoarthritis include the vitamin D receptor gene, insulin-like growth factor I genes, cartilage oligomeric protein genes, and the HLA region (Felson et al., 2000). Local mechanical factors include the body weight and the pathological alterations of the mechanical environment of the joint. Persons who are overweight have a high prevalence of knee osteoarthritis (Felson et al., 1997). OA is also considered to be related to alterations in joint mechanical environments such as knee laxity, the displacement or rotation of the tibia with respect to the femur; proprioception, the conscious and unconscious perception of joint position and movement; knee alignment , knee position in reference to the hip and ankle (Felson et al., 2000). In addition, joint dysplasias, fractures of articular surfaces, and tears of menisci and ligaments that increase joint instability precede the development of osteoarthritis in a high percentage of affected joints. Risk factors for posttraumatic osteoarthritis include high body mass, high level of activity, residual joint instability or malalignment, and persistent articular surface incongruity (Buckwalter et al., 1997; Honkonen 1995). 3.5 Treatments The medicine treatment of OA was dominated by COX2 inhibitors (Flower 2003). The other medicines include glucosamine, chondroitin (McAlindon et al., 2000), and hyaluronic acid (Lo et al., 2003). In addition, both aerobic walking and muscle strengthening exercise reduce pain and disability from osteoarthritis (Roddy et al., 2005). Articular cartilage lesions, both of traumatic or pathological origin, do not heal spontaneously and often undergo progressive degeneration towards osteoarthritis (OA). The most frequently used treatments include the artificial joint replacement, mosaicplasty, marrow stimulation, and autologous condrocyte implantation (ACI) (Steinwachs et al., 2008). Total joint replacement is most commonly performed in people over 60 years of age. (NHS 2006; Brittberg et al., 1994) Mosaicplasty is an autologous osteochondral transplantation method through which cylindrical periosteum grafts are taken from periphery of the patellofemoral area which bears less weight, and transplanted to defective areas. This transplantation can be done with various diameters of grafts (Haklar et al., 2008; NHS, 2006). Marrow stimulation methods include arthroscopic surgery to smooth the surface of the damaged cartilage area; microfracture, drilling, abrasion. All marrow stimulation methods base on the penetration of the subchondral bone plate at the bottom of the cartilage defect. The outflowing bone marrow blood contains the mesenchymal stem cells which are stabilised by the clot formation in the defect. These pluripotent stem cells which are able to differentiate into fibrochondrocytes, result in fibrocartilage repair with varying amounts of type I, II and III collagen (Steinwachs et al., 2008). The ACI tissue engineering treatment will be discussed in the next chapter. Chapter 4 Tissue engineering and autologous chondrocyte implantation (ACI) 4.1 Overview of tissue engineering technologies Tissue engineering is defined as ‘‘the application of the principles and methods of engineering and the life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve tissue function† (Langer and Vacanti, 1993). Three factors are considered as the principles of tissue engineering, including the utilization of biocompatible and mechanically suitable scaffolds, an appropriate cell source, and bioactive molecules to promote the differentiation and maturation of the cell type of interest (Song et al., 2004). Potential applications of tissue engineering are involved in the following fields: skin, cartilage, bone, cardiovascular diseases, organs (e.g. liver, pancreas, bladder, trachea and breast), central nervous system (e.g. spinal cord), and miscellaneous (e.g. soft tissue, ligaments). Although research is being carried out in all these fields, only few products have already entered the market. The most successful products up to now are: tissue engineered skin which is mainly used for wound cover, autologous chondrocyte implantation (ACI), and artificial bone graft (Hà ¼sing et al., 2003). 4.2 Autologous chondrocyte implantation (ACI) In 1984, a study in rabbits reported successful treatment of focal patellar defects with the use of ACI. One year after transplantation, newly formed cartilage-like tissue typically covered about 70 percent of the defect (Grande et al. 1989). In 1987, Brittberg firstly performed ACI in 23 people with deep cartilage defects in the knee. ACI is described as the following procedure: cartilage cells are taken from a minor load-bearing area on the upper medial femoral condyle of the damaged knee via an arthroscopic procedure, cultivated for four to six weeks in a laboratory and then, in open surgery, introduced back into the damaged area as a liquid or mesh-like transplant; at last, a periosteal flap sutured in place to secure the transplant (Figure 2; Brittberg et al., 1994). Genzyme Biosurgery with its product Carticel ® was the first company which introduced ACI into market and is the market leader in USA. Carticel ® is a classic ACI procedure using the periosteal cover (Hà ¼sing et al., 2008). Today the periosteum is often replaced by an artificial resorbable cover such as collagen I/III and hyaluronan membrane, such as ChondroGide or Restore (De Puy, Warzaw, Indiana) (Gooding et al., 2006; Jones and Peterson, 2006). Another new method uses chondrocytes cultured on a tri-dimensional (3D), biodegradable scaffold. This kind of scaffold, cut to the required size, is fixed into the lesion by anchoring stitches or its sticky nature. The 3D cell seeded scaffold eliminates the using of cover, thus simplifies the surgery procedure, saves the surgery time, and opens up the possibility of an arthroscopic surgery instead of the open surgery which causes more tissue damage. HYALOGRAFT from Italy is one of the European market leaders. It is a cartilage substit ute made of autologous chondrocytes delivered on a biocompatible 3D matrix, entirely composed of a derivative of hyaluronic acid (Marcacci et al. 2005). 4.3 Clinical results of ACI Brittberg studied the long-term durability of ACI-treated patients, 61 patients were followed for at least five years up to 11 years post-surgery (mean 7.4 years). After two years, 50 out of 61 patients were graded good-excellent. At the five to 11 years follow-up, 51 of the 61 were graded good-excellent (Brittberg et al., 2003). Since 1997 the year FDA approved ACI, this method has been widely performed in more than 20,000 patients all over the world. It has been reported to be effective in relieving clinical symptoms, such as pain and function (Wakitani et al., 2008). In a randomised controlled study, Knutsen et al. studied 80 patients who needed local cartilage repair with lesions on the femoral condyles of 2-10 cm2. There were no signi ¬Ã‚ cant differences in clinical results at 5 years follow-up (Knutsen et al., 2007). In another randomised controlled study that compared mosaicplasty with ACI, there was no significant difference in the number of patients who had an excellent or good clinical outcome at 1 year (69% [29/42] and 88% [51/58], respectively). In the subgroup of patients who had repairs to lesions of the medial femoral condyle, significantly more patients who had ACI had an excellent or good outcome (88% [21/24]) compared with those who had mosaicplasty (72% [21/29]) (p Clinical results of ACI were reviewed by Gikas 2009 (Gikas et al., 2009). Generally speaking, the outcomes of ACI treatment have been encouraging. However, most randomised controlled studies showed no significant difference between ACI and traditional treatments. 4.4 Limitations of ACI Microfracture is a very simple and low-cost procedure whereas ACI costs about $10 000 per patient. If ACI is not found to be more effective for improving articular cartilage repair than microfracture, the procedure will not be continued (Wakitani et al., 2008). There are several possible reasons to be blamed for the limitations of the traditional ACI procedure. The cell source in ACI is the cartilage tissue derived via an arthroscopic procedure from the low load-bearing area on the upper medial femoral condyle of the damaged knee. However, Wiseman et al. found the chondrocytes isolated from the low loaded area of the knee joint respond to mechanical stimulations in a distinct manner with the chondrocytes from the high loaded area, which suggests the traditional cell source of ACI may not provide enough mechanical response and may further lead to the insufficient mechanical properties of the repaired tissue (Wiseman et al. 2003). As cultured in monolayer, chondrocytes undergo a process of dedifferentiation and adopt a more  ¬Ã‚ broblast-like morphology, which is accompanied by an increase in proliferation and an altered phenotype. Type II collagen, the major protein produced by chondrocytes in articular cartilage, are down-regulated in the culture, while collagen types I and III are increased (Glowacki et al., 1983; Stocks et al., 2002; Benya et al., 1978). The agregating proteoglycan aggrecan of articular cartilage, is down-regulated during dedifferentiation and replaced by proteoglycans not speci ¬Ã‚ c to cartilage, such as versican (Glowacki et al., 1983; Stocks et al., 2002). Therefore, monolayer cultured chondrocytes do not express the origninal phenotype, and their ability to regenerate damaged cartilage tissue is impaired. Upon implantation, dedifferentiated cells may form a  ¬Ã‚ brous tissue expressing collagen type I that does not have appropriate mechanical properties, which may lead to deg radation and failure of the repair tissue (Brodkin et al., 2004). Chondrocytes grown in conditions that support their round shape, such as plating in high-density monolayer (Watt, 1988) and seeding in 3D structure (Benya and Shaffer, 1982) can maintain their differentiated phenotype much longer compared to cells spread in monolayer cultures. Although ACI can still be considered to be one of commonly form of repair of cartilage defects, it does have a number of scientific limitations. Some of those can be resolved using more comprehensive tissue engineered strategies which incorporates cells, scaffold materials and potentially biochemical, biomechanical and/or physical stimulation in a controlled bioreactor environment. 4.5 Tissue engineering strategies for ACI Chondrocytes derived from the low load bearing area of the knee joint respond in a distinct manner with the chondrocytes from the high loaded area. Chondrocytes cultured in monolayer have a dedifferentiation phenomenon as described above. In addition, the limitation of the transplant volume is always a major problem in autograft to be overcome (Kitaoka et al., 2001; Vinatier et. al, 2009). Accordingly, potential cell sources are widely studied for the future improvement of ACI approach, which will be discussed in Chapter 4. Seeding in 3D structures (Benya and Shaffer, 1982) can maintain chondrocytes differentiated phenotype. Ideally, cell scaffolds for tissue engineering should meet several design criteria: (1) The surface should permit cell adhension and growth, (2) neither the polymer nor its degradation products should provoke inflammation or toxicity when implanted in vivo, (3) the material should be reproducibly processable into three dimensional structures, (4) the porosity should be at least 90% in order to provide a high surface area for cell-polymer interactions, sufficient space of extracellular matrix regeneration, and minimal diffusional constraints during in vitro culture, (5) the scaffold should resorb once it has served its purpose of providing a template for the regenerating tissue, since foreign materials carry a permanent risk of inflammation, and (6) the scaffold degradation rate should be adjustable to match the rate of tissue regeneration by the cell type of interest (Freed et al., 1994). Synthetic materials such as poly (glycolic acid) (PGA), poly (lactic acid) (PLA), and poly (lactic-co-glycolic acid) (PLGA) have been investigated for use as cartilage tissue engineering scaffolds (Cima et al., 1991; Vacanti et al., 1991). Both, in vitro and in vivo studies have demonstrated these scaffold maintained the chondrocyte phenotype and the production of cartilage extracellular matrix (ECM) (Barnewitz et al., 2006; Kaps et al., 2006). Moreover, PLGA is used as a scaffold material for matrix-based autologous chondrocyte transplantation clinically (Ossendorf et al., 2007). Natural materials have also been investigated in the application of tissue engineering scaffolds in ACI. Collagen-based biomaterials are widely used in todays clinical practice (for example, haemostasis and cosmetic surgery). Collagen is also be commonly used as main components in tissue engineered skin products. Several commercial ACI products have used collagenous membraneas as the replacement for the periosteum to close the defect, such as ChondroGide or Restore (De Puy, Warzaw, Indiana) (Cicuttini et al., 1996; Jones and Peterson, 2006). The combination of type I collagen with GAG in scaffolds had a positive effect on chondrocyte phenotype (van Susante et al., 2001). Hyaluronic acid is a non-sulphated GAG that makes up a large proportion of cartilage extracellular matrix (Schulz and Bader, 2007). Matrices composed of hyaluronan have been frequently used as carriers for chondrocytes. Facchini et al. con ¬Ã‚ rms the hyaluronan derivative scaffold Hyaff  ®11 as a suitable scaff old both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects in their study (Facchini et al., 2006). Sugar-based natural polymers such as chitosan, alginate and agarose can be formulated as hydrogels and in some cases sponges or pads. Although these materials are extensively used in in vitro research, their role in in vivo cartilage reconstruction is still limited (Stoop, 2008). Growth factors are proved to be able to promote the formation of new cartilage tissue in both explants and engineered constructs. Insulin-like growth factor-I (IGF-I), transforming growth factor-ÃŽ ²1 (TGF-ÃŽ ²1) increases, basic fibroblast growth factor (bFGF) can stimulate cell proliferation and/or biosynthesis in chondrocytes which were