The Smile Factory: Work at Disneyland

The Smile Factory: Work at Disneyland SELF-EXPERIENCE ON DISNEYLAND In general, this case shows the power of culture. It dominates how the Disneyland uses the culture to manage the employees’ behavior in the part. So that they can produce the products, the joyful experience in the journey on Disneyland. My own experience to Disneyland is that it provides the Care, the Warm Welcome and the feel of being in a fancy world since you are entering into Disneyland. Or we might say, it’s anywhere and anytime.The Disneyland, it’s like a factory associated with standard procedure to provide a wonderful journey to its customers through different product lines. ANALYSIS 1. The key elements of Disney’s culture: In general, marshal the will to meet and perhaps exceed customer expectations. That is, providing smiles to customers who are visiting the park. And the most important is to let customers feel the smile provided by Disneyland. Disney enhances its culture through : )Pre-hired training in Disney University; On the job training and self-motivated, monitoring and helping each other on the daily work; b)Create a standard guideline for all employees to follow up and encourage a internal competition for a higher level symbolic position. Create a small society in the park and set up a work culture that leaves little room for individual experimentation. c)Not only to entertain customers but also to make its employee feel happy when working in Disneyland. )Though not all the employees can match the guideline of being nice but still it reach quite a good position to provide a good feel to customers. 2. The role that the culture plays in Disney’s success in satisfying their customers: The culture is like kind of compass. As Bill Ross, a Disneyland executive put it: â€Å"Although we focus our attention on profit and loss, day-in and day-out we can not lose sight of the fact that this is a feeling business and we make our profits from that. â₠¬  This make Disney to set up rocedure and guideline on each position very detailed. By do so, Disney will then able to satisfy their customers successfully. 3. I have worked with Maersk Line, a world’s leading container shipping company with its head office located in Copenhagen, Denmark. This company’s culture, the Blue Dragon is â€Å"cult-like† in the way described by Collins and Porras. This brings good as well as bad attitude to the organization. On one side it creates a strong cohesiveness in the organization.Everyone is proud to be one of the members of Maersk Line. They are thinking that their blood is BLUE. But on the other side it makes the employees of this company to be a bit arrogant to insiders as well as outsiders. It makes employees to compete with each other and over tough to outsiders such as customers and suppliers. The culture has brought a big trouble to Maersk Line during the financial crisis worldwide. That is not willing to change and s low in action. Finally Maersk Line made a huge loss.

Does Religion Cause War?

I. IntroductionSince writing has been invented about 5,200 years ago, there has been religion in some levels. Whether it is the belief to a Christian God, or whether goes further back in history to Hinduism or Judaism, Buddhism and along with other religious beliefs. During that same period of time, there have also been a large number of wars and battles between different countries and civilizations. But what are the causes of these wars? Many argue that such things as politics, power and material goods are the causes of these wars, whereas others claim that it is religion which is the main reason.Where it can be said that religion has had a large influence on many conflicts in the past, it can also be argued that political issues and power struggles have had just as large an impact. While some argue that religion is the cause of wars, it is inconclusive whether war would or would not exist if there was no such thing as religion.Based on our research, some would say The Crusades are a major example of religion causing war. From the 11th century to the 13th century Christian states in Europe launched what are sometimes called Holy Wars against Muslims in the East. These wars centered on the city of Jerusalem which held a Holy significance in the Christian world. However when 3000 Christians were massacred, this began the retaliation and therefore the beginning of The Crusades to release the Holy City of Jerusalem from the rule of Muslims. In this example, religion has clearly caused The Crusades as there would not have been any war if the city of Jerusalem did not have Christian significance.II. Counter ArgumentsHowever, a contrary argument to this would be that it was the massacre of 3000 Christians which caused The Crusades to begin and not because of differing religious beliefs, although it is unquestionable that religion played a major part in the beginning of the Crusades as the massacre may not have taken place without a religious background. As the argume nt for religion causing war heavily outweighs the opposing argument, in this context it is fair to say that using this example, religion does cause war.Although the previous example concerning The Crusades suggests that religion does cause war it would be unfair to generalize this conclusion from one example of war to war in general. There are many counter arguments when discussing the topic of religion causing war such as political or cultural issues. One such example of this is the conflict in Northern Ireland which is commonly perceived as a religious one although religious and political leaders have used religion to incite division or unison within their people.The Unionists (those who wish to remain a part of Great Britain) are predominantly Protestant whereas the Nationalists see themselves as Irish and are usually of the Catholic faith. It is this difference between the identities (British or Irish) of the people which has caused conflict and not because of the commonly held belief that it is one of religion and sectarianism. By using this example it is clear that religion is not the sole cause of war although it can be used as an incentive to continue a conflict which is shown through the conflict in Northern Ireland where it has almost become a religious conflict, such is the commonly held view of the hostility. This example can therefore be used as basis for an argument stating that religion does not, in general terms, cause war.III. Our ArgumentsClaim #1Ignoring The Crusades, and more specifically the First Crusade, it is difficult to confidently state that there have been other wars which have undeniable religious causes. However, one which has underlying holy origins is World War II. In the book Mein Kampf it states â€Å"hence today I believe that I am acting in accordance with the will of the Almighty Creator: by defending myself against the Jew, I am fighting for the work of the Lord† (Hitler, 1924) While some say that Hitler was irratio nal in his thinking, it  is undeniable that one of the main reasons for creating the Nazi Party was a misguided religious belief, such as the one above, and therefore the beginning of World War II can be put down to religion to an extent. This is shown by his actions following his writings in Mein Kampf.Claim #2To an extent, the â€Å"anti-terrorism† war in Afghanistan is also an example of a war with a religious cause although some argue that the war began for other reasons. As the terrorist attacks in America were the catalysts for war in Afghanistan, it can be said that the conflict did have a religious cause. This is because of the terrorists having a misguided belief, much like Hitler and his determination to eradicate the Jews, that they were acting in the name of an Islamic God. Because of this belief the cause of the conflict in Afghanistan can be put down to religion and therefore suggests that religion can cause war and conflict.Claim #3On the other hand the examp les of wars which have been caused by religion cannot automatically assume that all war is caused by religion or generalize a cause of one conflict to all conflicts. Following on from this another example of a war which does not have a religious cause is the Gulf War in 1990. The basis for war in this example was the invasion of Kuwait by Iraq, caused by a non-religious belief that Kuwait was a part of Iraq, along with the fact that Saddam Hussein wanted to invade and capture more land for himself. Therefore, this example again confirms the idea that religion is not the sole reason for war and that again, political issues are just as likely to be the catalyst for conflict.IV. ConclusionA final argument against the claim that religion causes war is one which is based on opinion and also has large quantities of evidence to back up the point; that war would occur anyway even if religion did not exist. One viewpoint is that it is human nature to always want more (greed) and this, more t han religion, can cause conflict with others which may lead to war. The evidence to support this point comes from the large number of wars and conflicts which do not have religious causes.In conclusion, it is unfair to suggest that there would be no war if religion did not exist as it is probable that human beings would use other motives for war or find other things to fight over. However, having said that it would also be unfair to declare that religion does not cause war as The Crusades, World War II and the war in Afghanistan to name just a few, defy that notion. Following on from this, some wars do have religious causes but consequently it is false to suggest that religion causes war in general as there are other hugely significant causes such as political issues, power struggles and the greed of humanity for more material possessions which are just as likely causes for war as religion.

A Framework Model for an Online Examination Timetable using Constraint Dissertation

A Framework Model for an Online Examination Timetable using Constraint Programming, PHP and MySQL - Dissertation Example The challenge and complexity of the problem lies in the fact that institutions may need to satisfy a set of constraints that might be too diverse or even contradictory. There are a few constraints that cannot be violated at all (hard constraints), few constraints are non universal (soft constraints) and may or may not be followed by an institute and lastly, there may be constraints unique to a specific institute (Burke et al. 1995). Problem Statement Academic institutions all over the world are required to go through the tedious and time consuming task of producing examination timetables periodically. Therefore, a universal solution for the examination timetabling problem would have a substantial impact factor. Owing to the fact that different institutes require a solution satisfying different constraints, the problem of finding a generalized solution that caters all these differences could be rather challenging. Devising a universal model for examination timetable problem would requ ire flexibility in terms of the specified constraints and commercial software cannot provide that. Aims and Objectives The aim of this project would be to suggest a universal framework model for the examination timetabling problem. A solution that ensures provision of flexibility in terms of constraint specifications shall be proposed. ... Literature includes timetabling systems presented by Hansen and Vidal (1995), Colijn and Layfield (1995), Lim et al (2000) and Dimopoulou and Miliotis (2001). Various approaches have been suggested by researchers and universities to solve the examination timetabling problem. Some survey papers have been published over time listing the techniques that have been utilized in addressing the exam timetabling problems. These include the survey by Carter and Laporte (1996), Burke and Petrovic (2002), Schaerf (1999), Petrovic and Burke (2004) and Burke et al. (1997). Amongst the approaches include methods based on evolutionary algorithms (Cote 2005), clustering, graph based sequential methods, case based reasoning (Gaspero & Schaerf 2001), hyper heuristics (Burke et al. 2007), harmony search algorithms (Burke et. al 2004), tabu search (Gendreau & Potvin 2005), particle swarm algorithms (Gaspero & Shuref 2001), and simulated annealing (Chiarandini 2006) have been proposed for the examination timetabling problem. It has been observed that hybrid methods in general give better solutions that pure algorithms. However, efficient integration is required rather than sequentially integrating the different approaches (Que et al. 2006). For building timetabling systems, researchers have used some general constraint programming packages e.g. ECLiPse (Ajili & Wallace 2003). A few efforts have been seen in literature for standardizing the modelling language and data format (Kingston 2001; Ozcan 2003; Reis & Oliveira 2001) once the need for it was recognized (Burke et al. 1998). Methodology The project has both research and development phases, so time shall be divided accordingly. The framework model would have the server-client architecture and would comprise of

Personal essay Example | Topics and Well Written Essays - 750 words - 1

Personal - Essay Example However, the two terms are quite distinctive from one another in the context of a number of differences such as the differences on the level of education and knowledge, responsibilities, and professional advancement. In terms of education, the difference between LPN and RN is quite evident - it is precisely described as the difference between diploma and degree in the respective manner. Now transitioning from LPN to RN definitely has better opportunities for career advancement along with added responsibilities. The latter is very significant that can be identified with the concept of nursing as a lifelong learning; because, â€Å"Nursing is a dynamic, continually evolving discipline that employs critical thinking to integrate increasingly complex knowledge, skills, and technologies and client care activities into evidence-based nursing practice.† (Harrington and Terry, 2009, p. 4) This paper briefly discusses the issue of transition from licensed practical nurses to registered nurses in conjunction with a number of aspects related to both terminologies as well as the transition phase. It also looks into significant issues relevant to LPN-RN transition including the analysis and evaluation of personal goals and expectations, educational as well as professional outlook, and the issue of career transformation with added professional and personal responsibility. With regard to choosing RN programs over LPN programs, licensed practical nurses are now more focused on continuing their education and becoming RNs. Apart from educational improvement and professional enhancement in general, the decision also includes the relevance of personal choices, goals and expectations in particular. With the difference of educational curriculum and learning styles, transition from LPN to RN also involves the aspect of role development. That is to say, the transition from practical nursing education to higher

Financial Reporting Disclosures in the Australian Corporate Sector Essay

Financial Reporting Disclosures in the Australian Corporate Sector - Essay Example More, the standards require that the identifiable intangible assets must be disclosed by means of subdividing the assets into classes with similar identifiable intangible assets being grouped together with regards to their usage and operations. Cash generating units impairment testing should be through the process of comparing the carrying amounts of identifiable intangible assets, goodwill working capital, and PPE of the cash-generating units. It is also important to note that valuation approaches during disclosure needs to be financial ratios and discounted cash flow. The company also needs to ensure that sensitivity analysis is done in case there are changes in key assumptions used during financial reporting. The importance of carrying out a sensitivity analysis to changes in key assumptions is due to the need to evaluate variations and their effects on financial reporting. Sensitivity analysis will also enable corrective actions to ensure compliance of financial reporting require ments. According to analysis of the company’s reporting practice, forecast must be corrected to be based on present value of future expected future cash flows. Forecasts on future cash flows must also be based on an established cyclic cash flow pattern. Table of Contents Introduction Identifiable Intangible Assets Impairment Testing on Cash Generating Units Calculation of Discount Rates and Growth Rates Sensitivity analysis to Key Assumption changes Financial Reporting Practice of CCA LTD Recommendations Financial Reporting Disclosures in the Australian Corporate Sector Introduction Corporate companies are subject to Corporations Act, which is being promoted by ASIC reviewers to ensure compliance with financial reporting requirements. The ASIC further provides non-compliance allowance for specific requirements to corporations. Confidence of investors and integrity levels in the Australian Corporate sector is largely boosted by the ASIC activities of monitoring corporationâ₠¬â„¢s compliance to financial reporting requirements. Users of financial reporting and auditing information are able to make informed decisions about the reliability and relevance of financial reporting disclosures in the Australian Corporate Sector. There is need for Coca Cola Amatil LTD to ensure that their financial reporting standards adhere to the professional and legal requirements of the corporations act. The objective of these financial reporting disclosures report is to ensure that Coca Cola Amatil LTD adheres to corporate Act’s requirements of financial reporting disclosures in the Australian Corporate Sector. Identifiable Intangible Assets The corporate act specifies reporting standards that must be adhered to when disclosing information regarding identifiable intangible assets such as names of brands, relationship with customers and written off intangible assets of Coca Cola Amatil LTD. The company should disclose identifiable intangible assets that would have bee n recognized during business formation or combinations and research and development assets. These intangible assets do not include assets that are recognized through contract basis or any other means that is legal. During financial reporti

Rag time (movie) Essay Example | Topics and Well Written Essays - 750 words

Rag time (movie) - Essay Example the height of success and sophistication (Forman, n.p.).Walker Coalhouse has walked up the ladder of the social class courtesy of his talent in music, where he plays the piano in a successful Jazz band, allowing him to become successful both in fame and fortune (Canby, n.p.). Under the current social stratification of the community in which Coalhouse lives, he belongs to the bottom layer of the social class; the blacks, and thus some of the white people volunteering at a firehouse in New York cannot stand to see a black man riding in such an expensive and state-of-the-art car. This makes the fire chief, Conklin to rally the volunteers to blockade the way and stop him from passing with his car, and later leads them in wrecking Coalhouse’s Model-T (Forman, n.p.). The attempt by Coalhouse to seek justice for the racial mistreatment that he received fails completely, because the justice system is dedicated to racism, thus it cannot offer a black man any justice (Forman, n.p.). Starting with the search for a lawyer to represent him in the case to petitioning the police to address his case by having the fire chief charged for the injustice he committed against him, the whole system refuses to work in his favor not because Coalhouse is on the wrong or because he cannot afford to pay the requisite legal fees, but simply because he s black (Canby, n.p.). Therefore, the theme of racism has featured in the entire film, building up from one episode to the other until the film ends without a clear solution to the problem of racism having been devised. The motif of the film is presented through fantasy and illusion, where the film has mixed up real life characters and events with other imaginary and unreal narratives that create a sense of confusion and lack of comprehension of the film. The historical events in the film are so mixed up, while there lacks the focus on one-sided historical narration (Canby, n.p.). Contrary, the film presents a multiplicity view of the

World War II through the 1970s Research Paper Example | Topics and Well Written Essays - 750 words

World War II through the 1970s - Research Paper Example The very first effect was the development of an anti-war attitude and a general decline in patriotism among a considerable number of Americans. The nation witnessed many protests like the Chicano Moratorium that marred the integrity of the nation. Since then, the nation has been ideologically split into two spheres in all military activities. Also, it becomes necessary for Presidents to select such generals and admirals who are ready to carry out the military policy laid out for them. Also, the Presidents never get the chance to get the people united even in war situations. The political impact of the war is evident from the fact that the War Power Resolution was enacted by the Congress to forbid the President from sending troops abroad without the consent of the Congress. According to Albert and Henderson (2002, 56-57), the war resulted in the incorporation of an anti-war sentiment and a Vietnam syndrome as evidenced by films, magazines and social movements. The Civil Rights Act too had profound impact on the American society, economy and culture. An immediate effect was the improvement in the education levels of the African American students. In the opinion of Donohue III and Heckman (1991), a similar trend was visible in gender equality too; and the legislation also resulted in a significant improvement in the average income of blacks. Also, it considerably improved the work environment for the immigrants. Thus, all these years, America remained an attractive destination for immigrants, which has both positive and negative consequences according to the scholars. Because of this race-friendly legislation, America has become a society with various races ranging from whites, Asians, American Indians, Hawaiians, and pacific islanders. This resulted in the development of a vibrant culture that has wide variety in music, literature and all other art forms. As the World War I ended, America realized the heavy loss it incurred as a result of the involvement in the w ar. Realizing the fact that the war was a disastrous mistake, the nation introduced a number of Neutrality Acts, and stayed away from issues like the Japan invasion of Manchuria, German effort to raise arms, and Italy’s invasion of Ethiopia. Secondly, America was facing the brunt of the Great Depression that had a traumatic effect on the nation’s economy. So, America was unable even to think about another war in the imminent future. Thirdly, America had peace treaties with a number of nations ranging from Britain, Japan and France. So, America was not willing to show more attachment towards any nation as it would dissatisfy the others. As Macdonald, Holden and Andener (1988) point out,women played a significant role in support of the American Military. Firstly, many women joined the nurse corps and the armed forces so that the men could be deployed in the combat front. Also, many women volunteered to run the government during the crisis. Secondly, females readily accep ted the traditional male jobs in factories when the factories were set for war production. Thirdly, women activists engaged in activities like social fundraising and blood collection for war (p.23). The first breakthrough (as cited in (Lawnix. Com, 1954), came in the year 1954 in the form of a Supreme Court ruling in Brown v. Board of Education of Topeka that setting separate educational facilities for blacks and whites is unconstitutional. It was this ruling that gave way to the civil rights movement. The second important breakthrough was the Voting Rights Act of 1965 that ensured voting rights for the blacks. As Ryan (2010) points out, the first political awareness came in the realization that America is fully of

Emerging New HR Concepts Essay Example | Topics and Well Written Essays - 2500 words

Emerging New HR Concepts - Essay Example The structure of an industry has a major role in defining a company's competitive advantage in this global scenario. An industry is conceived as a group of companies that market products which substitute each other. The structure of an industry has a direct relation to its profitability. Service industry can be considered as a classic example in this regard. In the post liberalization and globalization era, the concept of outsourcing has gained more attention. Cost is playing a major role deciding the competitive advantage of an organization. Companies are thinking of different options to reduce cost and increase its competitiveness. Outsourcing of production and service has been considered as an effective way to reduce competitiveness. Companies in the western world have been outsourcing jobs and production facilities from their land to low cost areas such as India, Brazil, China and East Asian countries. With the industrialization process of the 20th century, salary levels and capital cost of Western countries had shoot up substantially and this had been eating up a major pie from companies' profit. There was no sign of reduction of cost and salaries. This has compelled corporates to think of other options. During 1980's companies had find East Asian and Latin American countries as a good destination for moving production and services. While companies in Western countries have good technology, better expertise, capital strength and understanding of the market, these countries provide low cost land, human resource and raw materials. Synergising both these strengths has given companies more competitive advantage in their domestic market. Their production cost has come down sharply with this move. It is considered that high skilled labour, highly professional management team, low cost work environment, best management and HR practices, innovation, understanding of the industry and secu rity measures are the major strengths of the organization in the globalised world. Most of these strengths are adopted by companies do to the particular character of the outsourcing industry. In the early 1990's the industrial scenario in developing countries was quite different from that of western countries. Here companies were least bothered of good corporate governance practices, HR management, cost, profitability, security and customer satisfaction. The state protective measures made the industry inefficient. Companies got huge subsidies for every thing and any thing. But with the entry of multi national companies to these markets, situation has changed drastically and a new corporate governance practice has been established. In the labour relation also there has been a paradigm shift in the past one and half decade. Technology has played a major role in changing labour outlook and mindset. Also the process of Globalisation, Liberalisation and Privatisation has played a major role in redefining the perception on labor. Today corporates and industry are keener on optimum use of labor and they are always thinking of new ways to improve efficiency and output. The increasing global competition has demanded new approach to the labor. There has been an increasing demand for skilled labor forces. The skill level of the workforce in developing countries was a major challenge for companies moving from western market to emerging

Resource Concerns Research Paper Example | Topics and Well Written Essays - 750 words

Resource Concerns - Research Paper Example Resources as a Concern in Global Strategy Customers expect value for money when purchasing a product. The manufacturer has to ensure that the customer is delivered the greatest value for money while keeping base costs as low as possible. Globally, consumers tend to behave differently as per their geographical, cultural or other tastes. In certain cases, it is possible to substitute local solutions for customers but in other situations it is imperative to use original resources. The custom furniture manufacturing business under consideration is able to sell its products given their novel form. Such a form can only be maintained when using resources that were being used by the parent manufacturing plants located in the United States. However, it must be considered that importing such resources from the United States would cost heavily and carries the additional risk of making the products uncompetitive in an already competitive market. Resources of Concern in China China is a large buy ing and selling paradise for both consumers and manufacturers. Given that China produces a large number of manufactured products, it is highly likely that a number of critical resources might be available in China. However, certain specialized resources might not be available and might have to be outsourced from the United States before they can be manufactured locally in China. The custom furniture business relies in large part on the use of wood (Han, Wen, & Kant, 2009) as well as other construction materials such as wrought iron, stainless steel and novelty plastics. As far as materials such as wrought iron and stainless steel are concerned, China has an ample cheap supply of these materials. However, when the issue of woods and novelty plastics is considered, the situation is altogether different. Although China has an ample supply of wood but Chinese woods such as mahogany, oak wood or birch cannot be expected to provide the same output as local woods (Chunquan, Taylor, & Guoqi ang, 2004). Chinese focus lies more towards the production of synthetic boards to create furniture while custom furniture requires original wood for the exquisite feel and taste (Lee, 2011). The issue with novelty plastics is similar since they would have to be outsourced from the United States. In turn these imports would cost the company heavily and there may even be certain trade barriers for novelty plastics. On another note, China would provide a number of different textiles and fabrics for use in furniture manufacturing. However, in order to keep the look and feel of the original furniture, it would be necessary to import specialized fabrics. In the longer run, it may be possible to manufacture these textiles inside China but initially it would cost the company heavily. Impact on Decision to Expand in China Even though the Chinese market may hold some entry barriers such as the need to import certain resources but a competitive business can still be afforded given that Chinese imports of furniture originating from the United States totals some 8.8% of overall furniture imports (Aspin 2000, 2009). If the concerned company begins business in China by importing certain resources from the United States, the cost of the produced furniture would be lower than that of imported furniture. The lower cost of other base inputs such as electricity and labor would help to offset the cost of imports. Moreover, in the longer

Utility Programs and Macros Essay Example for Free

Utility Programs and Macros Essay Definition: A utility program is a program that performs a common task, such as copying and listing files on a list. Utility programs are generally provided alongside the operating system, but are sometimes bought separately. Many utility commands change with the operating system. Eg. In windows, the copy command is Ctrl + C, but in Mac OSx this changes to Command +C. The major tasks carried out by utility programs are: 1) Disk Formatting (Preparing either laser or hard discs for use) 2) Listing files in directories or folders. 3) Copying files, either from application to application, or within the same application. (Web browsers, word processors) 4) Copying discs 5) Renaming files 6) Backing up files, and also creating OS backups. (Time machine (Mac OSx), Windows Backup) 7) Running a file search in large files or directories Some other major tasks which operating systems often, but not always provide are: 1) File and Disc repair [Check disk, Defragmenter, Disk utility (Mac OSx)] 2) Anti-Virus tools, or malware checkers. (Windows Defender, Mac Cleaner) 3) Web Browser, to surf the internet. This could be standard, purchased, or freeware. (Safari, Internet Explorer) 4) File Compression, to compress files, and make the file size smaller. (Windows File Compression, Mac file Compression) Note: Often, these tools are not given by the Operating System, or are not advanced enough to be completely reliable. In this event, one may need to purchase the software. Eg. Norton Antivirus, Mcafee Antivirus, Mozilla Firefox, Google Chrome, Rockmelt, Winzip, 7-Zip, etc. MACROS: Definition: Macros allow you to automatically record a series of keystrokes, so that the key combination can record those keystrokes when required to do so. They are incredibly useful if you want to automate tasks which take too long to do, time and again, by hand. MACROS are available in most word processing applications, and are easy to use. They can save various things, such as fonts, font sizes, addresses, and even formats. One could create a MACRO that allows one to insert a document header, and footer, in a particular font, by just using two keystrokes. Creating a MACRO in MS Word is incredibly simple. This is how it is done: 1) Click on record MACRO at the bottom of the screen. 2) Give the MACRO a name. 3) Everything you do between turning the recorder on, and off, is recorded. 4) For example, you can insert a header and footer, type out an address, change the font to a particular size, etc. 5) In the status bar, click on stop recording. 6) In newer versions of Word, one must turn on the MACRO recorder in the developer tab, and then do what one wants.

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

American Needs a Medical School Application Loan Program Essay

American Needs a Medical School Application Loan Program For more than a year, a large part of my time has been spent applying to medical schools. I have dedicated much energy to prepare for and take the Medical College Admissions Test (MCAT), obtain multiple letters of recommendation from my professors, interview for a health professions committee letter of recommendation from my university, complete and submit the applications, and interview at various medical schools. Although this process proved to be a positive experience, the extraordinary cost of applying to medical schools poses a danger of limiting individuals with limited financial resources. Moreover, the lack of scholarship or loan programs to assist students with the application process further contributes to this danger. One of the first hurdles of applying to medical school is the MCAT. Although many students prepare for this exam on their own, a large portion of students choose to take preparatory classes offered by various private test preparation companies. These courses, which often cost in excess of $1000, teach students not only the basic concepts covered on the MCAT, but also helpful test-taking techniques unique to the MCAT. Thus, these preparatory classes may provide students with helpful advice and knowledge unavailable to those who cannot afford the classes. I attribute much of my success on the MCAT to these helpful hints; furthermore, many of my fellow pre-medical colleagues, who were unable to take the preparatory classes because of financial constraints, scored poorly on the exam. Hence, I believe MCAT preparation courses significantly increase one's probability of performing well on the MCAT, and, since these courses are out of reach for many... ...expenses should not impede a student's desire to apply to medical school and achieve his or her goal of becoming a physician. Although AMCAS and some medical schools have attempted to alleviate this problem by providing fee waivers or reductions, these efforts fall short of solving the problem of financial impedance to medical applicants. Many students from moderate income families cannot receive these fee waivers; moreover, their families cannot provide the $2000 to $3000 necessary to apply to medical school. Thus, I believe that the Department of Education, in conjunction with private loan companies, should provide low interest, medical school application loan programs. Through these loan programs, qualified students who lack necessary application funds, may rightfully apply to medical schools without facing the exuberant and potentially limiting application fees.