Clinical Experience With Urethral Reconstruction Using Tissue

Research Hypothesis

Describe  about the Clinical Experience With Urethral Reconstruction Using Tissue.

In context to the present day scenario, “three-dimensional tissue engineered models” of the “human oral mucosa” have been greatly developed , characterized and optimized that helped in understanding of the overall impact of the tissue engineering in the human beings. According to (Osman et al. 2015), various kinds of biomaterials are specifically used since the beginning of time as the connective tissue scaffolds that involved different types of cell culture techniques and other related tissue engineer approaches that helped in reforming the human oral mucosa (Barbagliand Lazzeri 2015). The important application of tissue-engineered mucosa primarily includes clinical transplantation and other related in-vitro investigations that helped in highlighting the investigation of the different kinds of interactions that lies in association with tissue engineered oral mucosa. The other related application included modeling of oral diseasing and further evaluation of the drug delivery system (Bhargava 2008).

In the coming years, the primary focus of the researchers has significantly highlighted on the optimization techniques that are used for defining the related models that specifically dealt with the “in-vitro and in-vivo” applications. Several studies based on the application of tissue based oral mucosa further helped in representing the capability of the use of advanced oral mucosal reconstructs that helps in reassembling the native oral mucosa (Chakrabarty et al. 2010). The reassembling of the native oral mucosa also helped in stimulating the clinical situation that lead to the reduction of the use of animal experiments. The recent advances in the field of tissue engineering involving oral mucosa thereby has a significant role in terms of the modern day experimentation that has been carried out in the field of biology (Bhargava 2008). This also includes the role played in the advancement, the application of tissue based modern oral mucosa, and how it remains related with the modern day application that specifically helped in better understanding of the knowledge that lied with the understanding of the application of the oral based tissue mucosa (Candiello 2010). Research based on the in vivo studies of animal has significantly helped the researchers to further investigate the importance of grafted tissue engineered oral mucosa in relation to healing of wound. (Patterson 2010), significantly evaluated the role of “autologous fibrin glue” based “engineered oral mucosa” by subcutaneous implantation that is specifically observe in case athymic mice. The major outcome of the of the study is that it clearly revealed that the epithelium remains viable and thus get expressed as a “cytokeratin profile”. The cytokeratin profile lies in close association with the native oral mucosa. Although it may become difficult for understanding and exploring the data associated with clinical situation particularly at places where the wounds are left open and the grafted material remains air exposed (Chappleand MacNeil 2012).

Research Aims and Objectives

It can be stated that the “grafted engineered oral mucosa” that is particularly based on the “human gingival keratinocytes” that are specifically cultured on a “fibroblast populated biodegradable collagen membrane on the dorsum of the athymic mice” (Chaudhuri 2011). Thus with the implementation of silicon transplantation chambers, the scientists were successful to protect the grafted materials. Histology based on the experimental report revealed that a well organized epithelium that has been found with no sign of necrosis (Colley 2011). Cultured keratinocytes were observed in studying the effects of implantation of the autologous canine oral keratinocytes. It was also observed that the cultured keratinocytes lost their activity on one week after grafting that particularly reported no improvement in the healing of the palatal wounds that helped the author to suggest that the “revascularization” of the injury area was very much slow (Fukahori et al. 2016). Hence, it did not allow the survival and the incorporation of the other substitutes that are used in the experiments. Although it was shown that the histological interpretation of the grafted engineered mucosa the cultured epithelium has peeled away and as a result the epithelial migration gets lost and peeled away (Gil et al. 2015)

The scanning microscopic studies mainly tended to show that various cells got enough growth and weredensely populated within the scaffold construction. The micro “Computed Tomography (micro CT) scanning” provided an increased porosity regarding the decrease of the total porosity situated at the end of the cultural period (Watkin et al. 2012). According to the histological examination protocol related to the oral mucosal model, it was clearly proved that there was a relative differentiation parakeratizedepithelium, which was found to be distributed evenly among the connective tissue layer (Goberdhan et al. 1993). Moreover, the tissue was found to be related to oral mucosal model as demonstrated by the researchers. The factor of feasibility related to the fabrication of a novel oral mucosal model mainly highlighted the cell lines. Thus, generating a “normal” cell models helped in optimizing the applications in both the in vitro and in vivo platforms (Harrison et al. 2005).

To find out if the vibrational spectroscopy will provide valuable information about the collagen cross linkage accruing in the “Contraction of Tissue Engineered Oral Mucosa”

• To analyze the degree of contraction and fibrosis of a “tissue-engineered oral mucosa” in vitro model
• Establishing a spectral profile related to the control along with the analysis of drug induced model in order to identify the key spectral differences that reflect on the biochemical changes within the collagen cross-linking.
• Monitoring the effects of B-APN on the drug induced models at different time intervals
• Constructing the chemo metric modeling system in order to analyze the spectral data

Tissue engineering is the most recent and appropriate application of biotechnology that has upgraded the way we can treat diseases and make innovations. One of the greatest application of this tissue engineering is the three dimensional reconstruction of the oral mucosa. This is helpful on cases of intra-oral and extra-oral repair and treatment of the defects associated with the soft tissues. In such cases it can be referred to as a graft material.  The reconstructions provide many useful advantages.  The three dimensional oral mucosa reconstructs is helpful in assessing the compatibility of dental implant and other materials. The three dimensional model also replaces the previously used animal model which was used to detect and analyze diseases like cancer ad pneumonia. Therefore the allocation of this tissue engineered oral mucosa tissue constructs can be used to detect diseases, assess the compatibility of the implants and other dental material, and also comes as an alternative to the previously used animal models (Moharamzadeh 2012).

Literature Review

Methods for reducing or inhibiting the contraction of the graft were analyzed and experimental design was set up to assess the efficacy of the methods used. The methods were preclinical in general.  The tissue engineered oral mucosa has previously shown to be helpful in treating the urethral stricture. Therefore studies were conducted to check which methods reduce the contraction of the graft. Using the tissue-engineered buccal mucosa (TEBM) method, three possible methods reducing the contraction was analyzed in vitro.  The methods investigated were “pretreatment of de-epidermised dermis (DED) with glutaraldehyde, culture with β-aminopropionitrile (β-APN; a lysyl oxidase inhibitor), and physical restraint of TEBM grafts during culture.”  Serial digital image analysis was used to assess the contraction and how it is influenced under the application of each of these methods. To conclude restraint of TEBM grafts during culture and Glutaraldehyde pretreatment were seen to reduce the contraction (Patterson JM 2016). The oral mucosa is the mucosa membrane that consists of stratified quamous epithelial cells. The oral mucosa is essential for protection, thermal regulation sensation and many more. Oral mucosa fibrosis is a condition of the mouth that becomes cancerous rarely. The condition is characterized by the juxta-positioning of the epithelial cells in the mucosa membrane. The condition gets worse by the inability of the patient to move or even open his/her jaws. The conditions of fibrosis can be treated with medicines.

“Tissue engineered oral mucosal equivalents” are been found to be developed for the clinical application and also for the in vitro studies related to “mucosal irritation disease, biocompatibility, and the other related basic oral biological phenomenon” (Candiello et al. 2013). Researches based on the reviews provide wide range of angles related to the tissue engineering strategies, which are related to the production of human oral mucosal equivalents (Harrison 2006).

Figure 1: Macroscopic view of co-culture system of Tissue Engineered Oral Mucosa

(Source: Barbagli and Lazzeri 2015)

The relative advantages along with the disadvantages are been systematically analyzed by the researchers related to the article. Numerous techniques, which are generally used for the in vitro reconstructions, associated to the human oral mucosa are been systematically analyzed by various researchers (Heller et al. 2016). This model generally helps the researcher in analyzing the concept regarding the advantages and disadvantages regarding in vitro reconstructions. This can be helped in analyzing clinical application at a better effect.

Figure 2: Histological Sections of Oral Mucosa Biopsy

Tissue engineered oral mucosa

(Source: Barbagli 2013)

Oral buccal (compositions may be often formulated in the form of tablet granules or capsule. However, the most preferable form is the oral buccal tablet. Due to the factor of high permeability along with effective oral absorption route, it can target in producing a rapid onset of the action of the drug within a very short delivery period (Mangera et al. 2013). Most of the researchers examining the concept of ““Contraction of Tissue Engineered Oral Mucosa””state that ageing is one of the most effective parameter, which affects the entire mechanism vastly (Kinikoglu et al. 2015).

Figure 3: A hematoxyline and esion stained of the tissue engineered oral mucosa equivalent consists of a well stratifies squamous epithelial layer

(Source: Bhargava 2008)

The lamina propia is considers to be fibrous along with highly vascularized layer, which is generally found to be situated underneath the epithelium layer. This is considered to e one of the most crucial factor for the nutrient supply associated to the epithelium layer (Neel et al. 2013). Since the success of the operative urethra reconstruction is determined by the size of adequate donor tissue for the tissue transfer, it can be considered as one of the major issue associated to the availability of limited quantity of donor tissues (Lee et al. 2015).

Schematic illustration mainly showing the methodology to produce “full thickness tissue engineered oral mucosal models”. In order to produce “tissue engineered normal oral mucosa model (TENOM), normal oral fibroblasts (OFs) along with normal oral keratinocytes (NOKs)” were seeded into a “DED scaffold” within a 0.8 cm2 steel ring (Moharamzadeh 2012). After the period of 72 hours, the composites were raised mainly to an air or liquid interface and thereby cultured for a further period of 14 days (Maeshima 2000)

The entire protocol for deducing full thickness “tissue engineered oral mucosa” has been summarized by the researcher in the proceeding page. As observed in all the three protocol, the primary incubation period was observed to be 72 hours. After plating on the secondary phase, i.e. the implementations of the media supporting the mimic cells are initiated. It can be clearly analyzed that the researcher provided sufficient period in the secondary phase in order to observe the differentiation.

In the secondary phase, the researcher allotted a total time of 14 days. However, there were much difference in the second experiment setup. It was observed that the researcher have analyzed for the implementation of Cal27 molecules during the primary phase. Moreover, in the secondary phase, the there were two systems which was analyzed. The first time setup was 14 days. However, the other period was much longer, i.e. 21 days.

Methods to reduce the contraction

After these two phases, the researcher successfully derived the “full thickness tissue engineered oral mucosal model”, which was the prime objective of the research study. After attaining this mimic model, various supporting experiment setup was organized in order to monitor the exact configuration of in vivo cell lines in oral mucosa of an individual.

Figure 4: Protocol for the production of “full thickness tissue engineered oral mucosal model”

In order to recreate an in vivo model of the “dysplastic oral mucosa (TEDOM)”, along with the “early invasive oral carcinoma (TEIOC)”, the “NOKs” were effective replaced with the OSCC cells (Cal 27 or the D 20) and thereby cultured for either a period of 14 days (2 weeks) or 21 days (3 weeks) respectively (Mangeraand Chapple 2013). In addition of a “MCTS to the TENOM” produced by a model closely, it can be observed that the entire system tends to mimic the carcinoma structure in situ (TECIS).

The mimic model model plays an important role for this research study. The replica would provide a complete identical environment of the composition, which is as found in vivo of human oral mucosa. Thus, any changes or alterations within the mimic model would directly reflect on the consequence of the actual in vivo protocol. Thus, effective strategies can be implemented in order to monitor the consequences in the model.

Introduction to Research Methodology

Research Methodology mainly helped the researcher in defining the most appropriate and suitable research approaches which in needed to adopt a complete result of the entire protocol related to the subject. This chapter provided a deeper and effective analysis of the factors involved in “Contraction of Tissue Engineered Oral Mucosa”(Mangera et al. 2013). It can be estimated that research methodology provided a better process for highlighting the limits associated to the protocol.

The research approach for a study is effectively required in order to format the requirements related to the study. The particular research study can by synthesized by two distinct ways, i.e. Inductive and Deductive approaches (Moharamzadeh et al. 2012). The inductive approach mainly targets in analyzing the research topic when there is limited data present for the relevant context. The preliminary phase of the inductive approach targets in observing relevant information and thereby building the research study systematically (Muzzarelli et al. 2015). It can be clearly noted that the deductive research approach can be used as a protocol to describe the practical application regarding the theories needed to access the content of the research study. On the other hand, the deductive approach targets in building theories related to concepts that are more specific associated to the data analysis structure. Thus, depending more on the nature of the study related to the research topic, the selection of an effective research approach are made to provide a better analysis of the concept (Nguyen 2012).

The present research study mainly focuses to the concepts related to “Contraction of Tissue Engineered Oral Mucosa” based on the theoretical knowledge. The various models related to the study helped the researcher to understand the subject more precisely and systematically. The deductive approach failed to implement effective concepts related to the present context. This is because the researcher heavily emphasized on the development of innovative concepts and ideas regarding tissue-engineered models (Harrison et al. 2005). On the other hand, deductive approach was found to be unsuitable for the study becausethe researcher emphasized more on the innovation of new concepts and not on developing theories based on the previous research studies of “Contraction of Tissue Engineered Oral Mucosa”.

The research design helped the researcher in explaining the framework related to the research topic. An efficient design helped the researcher in collecting data through a systematic and scientific manner (Ohki et al. 2012). The types of Research Design which are found to be associated to the study are as follows:

Figure 5: Research Design

(Source: Chappleand MacNeil 2012)

The Exploratory Research Design helped the researcher in acknowledging the vivid types of thoughts and ideas related to the context (Osman et al. 2012). On the other hand, the Explanatory Research Design mainly focused to towards the occurrence of various events or incidents, which affects the various consequences. The mere explanation regarding the cause and effect relationship limits the role of Explanatory Research Design to be applied in this context. Finally, the factor of Descriptive Research Study targets in gaining effective state occurrences related to the events in a proper and detailed manner related to the subject (Patterson 2011).

The application for the Explanatory Research Design mainly avoided the support regarding the concept of “longitudinal study”, which was possible for the particular topic. The “descriptive research design” was quite effective in defining the details of the various protocol related to the involvement of models for “Contraction of Tissue Engineered Oral Mucosa”(Roman et al. 2014). It can be considered apt for scrutinizing the research topic in a systematic manner.

Data are generally considered as one of the most useful protocol for collecting information and thereby analyzing factual information related to any research study (Vaegler et al. 2015). An effective and methodical data collection procedure would help in devising an accurate result for the research study.

The sources of data were found to be helpful for the researcher for analyzing the research topic and thereby extracting information in order to proceed accordingly (Watkin 2008). The Primary and Secondary Data sources were the two efficient bases, which helped the researcher in gaining profuse information for the research study. It can be stated that the primary data attained by the researcher mainly helped in collecting the raw information (raw data) which can be catered as per the needs of the research article (Winterroth et al. 2014). All the first hand data attained from the research survey needed efficient clarification on the fields of validity and reliability. On the other hand, the secondary data sources provided data, which mainly targeted for widening the concepts related to the research topic. This helped the researcher to analyze a better study of the subject as it provided in depth data and description (Xie et al. 2014).

The current research topic focuses on the primary research subjects, which effectively included the interactive sessions with scholars performing research on ““Contraction of Tissue Engineered Oral Mucosa””(Kinikoglu, Damourand Hasirci 2015). Apart from this, the researcher stressed on the literature sources of the dissertation (as it was considered as an output of the information attained from the secondary tools). The most efficient secondary tools, which were related to the dissertation, are offline and online platforms, such as the peer reviewed journals, blogs, websites and articles related to ““Contraction of Tissue Engineered Oral Mucosa””(Heller et al. 2016).

Qualitative Analysis

The Qualitative Data Analysis were considered as one of the most efficient parameter of the research study which helped to provide a better description of the research study. The study of qualitative analysis provided a practical application of the subject, which in turn helped in understanding the topic more precisely unlike the quantitative procedure (Kinikoglu, Damourand Hasirci 2015). The study was mainly based on non-probability sampling, because the entire data were restored and left non-manipulated.

The Quantitative Data mainly applied statistical data related to “Contraction of Tissue Engineered Oral Mucosa”, which was useful in recording data. The tools, which were implemented in the quantitative research study, were regression plot, ANOVA, T-test and Z-test in order to extract the information from large sample population (Lee et al. 2015). The study was mainly based on probability sampling in order to maximize the output attained from the entire methodological protocol.

In this present research study, the researcher has opted for a mixed analysis (i.e. the intervention of both qualitative and quantitative research study). This is mainly because the researcher wanted to extract data from bulk population along with quality information from the scholars undergoing researches on ““Contraction of Tissue Engineered Oral Mucosa””(Maeshima 2000).

The population related to the research study focused to the people who were either directly or indirectly associated to the research study. The present study mainly dealt with scholars related to the project of ““Contraction of Tissue Engineered Oral Mucosa””and the students studying under the same group of scholars (Mangera 2013). However, it was quite impossible for the researcher to extract information from every student, as the population size was huge. Hence, the researcher opted for the synthesis of a research sample, which would provide the exact replica of the entire population

The sample, which was considered for the study of “Contraction of Tissue Engineered Oral Mucosa”, was much smaller in number. The samples for the students were based on “simple random probability sampling”, where no criteria were selected. On the other hand, the scholars related to the article were based on the non-probability sampling technique. This was performed based on the online questionnaire template. The students were asked to participate in the survey, which was performed by the researcher based on the Liker’sScale (which ranged from 1 to 5). On the other hand, the scholars were accessed for the interview session as per the flexible timings and schedule.

The sample size of the research study was mainly based on the quantitative and qualitative research study. The study of the quantitative research sample was involved with 50 students who directly interacted with the research protocol. They were mainly provided with online questionnaire templates by the researcher. On the other hand, the scholars associated to the research topic of ““Contraction of Tissue Engineered Oral Mucosa””were 5 in number. They were conducted for an interview session. Thus, the total sample size (quantitative analysis of 55 + qualitative analysis of 5) were 60.

During the entire research protocol, the researcher were abided with various code of conducts, which helped in identifying the inappropriate measures regarding the research subject. The researcher was accustomed to wide range of decision making protocol in order to follow the ethical considerations related to the standardization of the research topic (Moharamzadeh et al. 2012). The commonly followed ethical considerations followed by the researcher are stated below:

Data Application: The data, which is gained from the study related to “Contraction of Tissue Engineered Oral Mucosa”, helped in better understanding of the subject. However, it was sincerely stated that the researcher did not followed any commercially available data available in the offline and online sources. All the data associated to the online and offline portal were considered as a backup of the secondary sources and was not directly incorporated in the project.

Respondents’ Involvement: The researcher stressed effectively on the fact that there was no external influence on the respondents regarding the participation in the study. Every participants involved in the study were voluntarily involved in the research study and had the liberty to step out from the program at any point of time.

Respondents’ Anonymity: The researcher ensured the fact that there were no factor of mental or physical harassment which were found to be involved with the respondents. Moreover, the personal identities of the respondents were not disclosed (confidential) by the researcher.

The research topics followed by the researcher were based on cross sectional and longitudinal platforms, which mainly depended on the information available on the particular topic. The present research studies were mainly based on cross sectional study due to the fact that the researcher were subjected to various time constraints. The researcher in order to segregate the tasks in a detailed structure and thereby help in completing of the research work provided a Gantt chart.

The entire details related to the article are summarized below:

Main Activities/ Stages	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9
Oral Biopsy
Isolation
Cell Expansion (passage) and De-EpidermalizedDermisprepatation (DED)
Seeding on DED
Tissue Engineered Buccal Mucosa (TEBM)
Tissue Processing
Analyzing with FTIR
Analysis & Interpretation of Data Collection
Findings of the Data
Conclusion of the Study
Formation of Rough Draft
Submission of Final Work
TOTAL

The purpose of developing “full thickness tissue engineered oral mucosal model” was based on the mimic structure of the actual cell lines. The model provided the exact structure, which is normally present within the individual. Thus, the researchers can effectively encounter any information regarding the change or alteration in the cell lines due to the application of any foreign material. One of the most effective perspectives, which can be attained from “full thickness tissue engineered oral mucosal model”, is the effects or side effects of drugs on the cell lines. After the development of any innovative formulation of a drug, the mimic model can be sufficiently used to monitor its mode of action. It would help in analyzing the accurate dosage of the drug and highlight to the side effects. Moreover, it would also help in analyzing the factors for the development of alternative drug lines that are apt for oral mucosal cell lines.

Apart from the factor of drug test, the second most important factor, which can be analyzed from “full thickness tissue engineered oral mucosal model”, is the factor for development of cancer. Often it has been stated that studying the development of cancer cells or studying the proliferation of cancer cells in normal context is quite a complex task. However, if a researcher is subjected to “full thickness tissue engineered oral mucosal model”, then he/she can easily access the possibilities for developing cancer cells. This would help in analyzing the exact morphology of these cultivated cells, which can be restored for future usage.

The third scope for the usage of “full thickness tissue engineered oral mucosal model” is the development of spot thickness skin grafts. Previously, it was stated that there were several disadvantages for harvesting “full thickness tissue engineered oral mucosal model” for analyzing the marked pain situated at the palatal donor site. Hence, as a result of fact, most of the materials were found to be used after the major trauma, maxillofacial preprosthetic surgery and surgical restrictions. It was previously considered that anatomical along with biochemical similarities related to the mucosal cell lines are quite commonly available for using on large thicken skin defects. However, based on future studies, one can effectively design “full thickness tissue engineered oral mucosal model” for intraoral grafting which was lagged for technical purposes previously. Very few reports based on this factors can be introduced for developing wide range of “full thickness tissue engineered oral mucosal model”s which can be effectively used for the grafting purposes.

“Full thickness tissue engineered oral mucosal model” can be used for developing primary human oral keratinocytes, which can be effectively harvested from the palatal keratinized mucosa. The material can be effectively spreaded across the palatal keratinized mucosal cells in order to provide an environment, which is completely free from serum, transformed irradiated xenogenic feeder cells along with the interaction of pituitary extracts in a well defined culture medium. As soon as the sufficient oral keratinocytes are produced, the researchers can seed them into AlloDerm, which is an acellular dermal matrix (branded under the LifeCell). The entire protocol for harvesting the tissue can be used for maintaining a suitable intraoral grafting. An effective “full thickness tissue engineered oral mucosal model” would help in developing Good Manufacturing Practices (GMP) standards. This would help in ensuring fabrication of the consistent guidelines for the various cell based products, which are based on the “Centre for Biologics Evaluation and Research (CEBR)” along with the interaction of the “United States Food and Drug Administration (FDA)”. The GMP regulation would help to maintain an quality aporoach in ordr to manufacture instances related to the contamination and errors. The commonly observable factors related to this domain would include personnel qualification, record keepings, protocol validation along with complaint handling.

Evaluating the safety factors related to “full thickness tissue engineered oral mucosal model” would help the researcher to formulate an intraoral grafting protocol. The secondary objective, which would be analyzed by the researcher, would be to assess the efficiency of the grafted “full thickness tissue engineered oral mucosal model” in order to increase the amount of keratinized mucosa after the grafting protocol. The factor facilitating clinical trial would help in developing Independent New Drug (IND) application, which is an important tool for the future studies.

The present chapter mainly described the tools associated to the research study. It helped to highlight the protocols, which are found to be related to systematic extraction and analysis of the data (Moharamzadeh et al. 2012). The available research techniques were found to be related to be related to research methodology. The tools used in this study were sincerely stated to analyze “Contraction of Tissue Engineered Oral Mucosa” in a much processed and effective manner.

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