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Tissue Engineering: Fertility Preservation

University: De Montfort University Leicester

  • Unit No: 7
  • Level: High school
  • Pages: 27 / Words 6805
  • Paper Type: Essay
  • Course Code: N/A
  • Downloads: 545
Question :

This sample will let you know about the:

  • Discuss about the biomaterial for fertility preservation.
  • Discuss about the Follicle and oocyte measurement.
Answer :
Organization Selected : N/A

1. Describe, Explain, clarifying & Summary for the following:

Article 1 Ahmed Baker and et.al., Characterization of decellularized and recellularized mouse ovary for future reproductive bio-engineering application

Material & methods

Scaffold generation from isolated mouse ovaries

The researcher conducted oophorectomy on female mouse that was aged between 10- 20 weeks. Here, the work was approved by animal committee of University of Gothenburg. The ovaries were dissected free from tissue and put into perafadex and then stored at -20 degree Celsius after processing it further. Then, ovaries were divided into groups and one of it was selected for decellularization protocol for 10 hours that consists of 0.5% of sodium dodecyl sulfate, for 16 hours in sodium deoxycholate. Moreover, in combination of two protocols SDS for 5 hours followed by 2% and SDC for 8 hours. Then, ovaries were washed with water and was kept in DNase I solution for 30 minutes at 37 degree Celsius. So, this process was followed with all ovaries. They were sterilized in peracetic acid for 30 mint. However, for the study ovaries kept in perfadex were taken to compare it with normal tissue.

Moreover, ovaries were fixed in formaldehyde for 1 hour, dehydrated and fixed in paraffin. Now, 5 µm section were cut then rehydrate. The staining was done of Hematoxylin and eosin with mixture of alcian blue. Further, immunohisto chemistry was done and ovary was exposed with citric acid in pressure cooker. For labelling cells, red fluorescent protein was applied. The strained section was visualized with leica microscope. Along with it, normal mouse ovaries were scanned using electron microscopy. After that, sample were mounted with 4nm gold particle.

The quantitative calculation was done using kits from Biocolor. For that total 6 ovaries were used and ECM analysis was done. Also, 12 decellularized ovaries of per group were analyzed as well. The Scaffolf toxicity test was conducted on tissues using 3 -2 -5 diphenyltetrazolium bromide. In addition to it, Roche's cell proliferation kit was used for analysis. A solution which included 20% SDS was used for death control of cells. Now, incubation of 8 hours 10 ul of MTT was added to culture cells. The active cells were converted into purple crystals and that was measured in reader of 565 nm. However, for recellularization each ovarian cell was scaffold using 30G needle. Then, it was cultured for 14 days then washed in PBS. In addition, total counting area was measured along with number of cells per square mm.

The statistical tests were conducted using Shapiro wilk test. In order to find out significant different in value Kruskal Wallis and Dunn test were conducted. The data was presented with median and range of 10 -90 %. Need Assignment Examples?Talk to our Experts!

Results & summary

It is analyzed that the methods of decellularization was effective. However, the mean elastin content observed in ovary tissue is 60% as compared with original ovarian elastic before decellularization. But the reduction was not normal in control tissue as it contained many variations within samples. Furthermore, in P1 treatment it was high decrease in sGAG's and collagen content. Thus, soluble and insoluble collagen was impacted in negative way. Also, it is evaluated that ECM macromolecules decreased in decellularization process by comparing with untreated mouse ovary. In MTT no cytotoxic remnants were included in scaffold type.

In RFP testing it was identified that all detected cells originated from recellularization. So, the cortex of ovary was properly recellularized in scaffold type as compared to ovarian medulla region. Furthermore, it is analyzed that high topography of recellularized cell using SEM reflected that the remaining ECM structure repopulated on surface of scaffold. But it was difficult to visualize cells in cross sectioned scaffold using SEM.

However, there is no difference between three recellularized ovarian scaffold types. But it was observed that there was large group variance. One ovarium of P2 category recolonized with average of 1277 cells/mm and in P1 scaffold it was 916 cells/mm and in P3 it was 541 cells mm. Moreover, there was no difference found in cell density of recelullarized scaffold in each group.

From figure 1 it is determined that no dark stained nuclei is found in decellularized ovary of scaffold. Also, use of DAPI ensured that DNS strain was removed after decellularization. From figure 2 it is identified that toxicity test removed toxic remnants from decellularization process. So, this indicated that scaffolds were non toxic. It is analyzed from figure 3 that scaffold cortex included more cells in central region. Moreover, the cells originated from recellularization process and not from incomplete decellularization. Alongside, it is observed that scaffold surface include large number of cells that confirmed ummunocyto chemistry and electron microscopy. There was presence of fluorescence in ovarian tissue as it is clearly scene in images of ECM structure.

Article 2 - M. C. Chiti: Fibrin in reproductive tissue engineering. A review on its application as biomaterial for fertility preservation

Material and method

In order to gather data and information about tissue engineering the methods used was secondary. For that secondary data was gathered through previous articles, journals, research paper, etc. here, an extensive medline search was conducted with help of different keywords. They were ovarian tissue, encapsulation, etc. thus, only English articles were selected and data was analyzed from it.

Result

It is concluded that in vitro study follicles grow to a soft part. The fibrin alginate gelation rate was faster in thrombin concentration and there was no difference in matrix rigidity of 3 combination. The follicles encapsulated was no longer embedded after 6 days of IVC. But 12 days of IVC there was clear evidence of follicle in fibrin matrix. However, combination of fibrin and alginate tunable degradability and mechanical property was positive and influenced follicle growth. Although follicle growth did not differ between matrices, oocytes were able to resume meiosis only in the latter hydrogel. The scaffolds produced same follicle rate and after 12 days it was double in size in fibrin as compared to 60% rise in diameter in alginate.

In vivo studies it is summarized that large follicles are lost in initial first day of avascular transplantation due to hypoxia. VEGF is released during fibrin HBP VEGF degradation increase revascularization. The first pregnancy occurred in fibrin HBP VEGF group. There was physical connection in grafted tissue and recipient. surrounding tissue. The suitability and safety of this procedure were further confirmed by another study, in which up to 100 leukemic cells encapsulated in an artificial ovary prototype did not induce leukemia after long-term xenografting, constituting one more key step towards clinical translation. Moreover, the presence of antral follicles and a new functional vasculature observed already 7 days after transplantation demonstrated, for the  time, the feasibility of constructing an ovary-like structure using fibrin as a temporary scaffold to encapsulate isolated follicles and ovarian stromal cells.

In transplantable ovary the presence of a basal membrane which encloses follicles, these entities can be mechanically and/or enzymatically isolated from the surrounding ovarian stroma. The leukemia was not induced even after using 100 leukemic cells into artificial ovary after long xenografting. It is analyzed that there is no difference in follicle apoptosis, growth and recover rates in F12.5 \T1 and F25 \T4. But the recovery rate are high in previous test within same group. The antral follicles were already present in vasculature within 7 days. Also, only a small dose of PL has positive impact on survival of follicle after grafting. But as 20% of fibrin was included the antral follicle was observed. Moreover, 48% survival rate of follicle was found in 15% PL fibrin. This was because of fast and effective vascularization. After 7 days of transplantation, both follicle groups were viable and growing, but primordial-primary follicles showed a lower survival rate that is 6% than secondary one that is 28%. Hence, primary follicle was influenced in similar way by isolation and grafting done. However, after grafting follicle had normal ultra structure and natural present in ovary. In secondary group the vascularization rate as high as compared to primary one. The artificial ovary containing more primordial follicle is able to store endocrine. Ask for assignment help from our experts!

Article 3 XU and et.al., Tissue engineered follicles produce live, fertile offspring

The material and methods followed in it are as follows :-

Animals

Here, immature follicles 16 day old female prepubertal were taken. Sperm was taken from CD1 male breeders. 8 -10 days week old mice was mated to vasectomized CD 1 for IVF. Then, animal were kept in room temperature. They were given food and water and libitum. The food given did not include soyabean and alfalfa meal.

Alginate hydrogel preparation

The sodium alginate containing 55- 65% guluronic acid was taken. The alginitic was dissolved into water of concentration level 1 % and purified with charcoal to remove organic impurities. Then solution were filtered through 0.22 um in conical tubes. Moreover, aliquot's of charcoal stripped and sodium alginate were reconstituted with sterile 1x phosphate buffered saline.

Follicle isolation, encapsulation and culture

The secondary follicles were isolated of mice and encapsulated into sterile of 1.5 % alginate. The ovaries were cubated into MEM containing 1% fetal calf serum, 0.1% type 1 of collagenase and 0.02% of D nase 1 at 37 degree Celsius and 5 % carbon dioxide for time of 30 minutes. After that follicles were isolated using insulin that contain 1% of FCS. However, the individual follicles were kept in MEM 1% FCS at 37 degree Celsius and 5 % CO2 for 2 hours before encapsulating them. Then, follicles that showed characteristics of 2 hours preincubation were selected. They were washed with 1.5 % of alginate two times. The single was washed only once suspending into a polypropylene mesh. It was then sterile into 50 mM calcium chloride for 2 min. the culture media included MEM 10 mIU/ml hormone, 3 mg/ml bovine serum albumin, 1mg/ml bovine fetium, 5 mg/ml insulin. The beads were plated in 100 ul of culture media. Now, follicles were kept at 37 room temperature and at PH of 7.0.

Furthermore, it was kept for 8 days. So, each day half media was stored in -80 degree Celsius. The follicle diameter was assessed in inverted lecia DM IRB through microscope. Those who wad dead if oocyte was not enclosed with granulosa cell. Then, culture media was replaced with 100 uL contained 10 mIU\ML alginate lyase for 30 min. Afterwards follicle were removed from bead using an IVF dish with 1% FCS.

Follicle and oocyte measurement

The pictures of encapsulated follicles were taken of 0, 4 and 8 days via a microscope. The diameter of follicles which were not matured was measured. However, diameter of follicles cultured in vitro were obtained on day 0 and 8 and then compared with vivo oocytes gathered from unexpanded cumulus oocyte of antral follicles of 24 days mice. The control oocytes were stripped by aspiration via glass pipettes. At last the diameter was measured without zone pellucida.

Oocyte maturation

The follicles were transferred to maturation media that was composed of MEM, 10% FCS, 1.5 IU\ml human chorionic gonadotropin, 5 ng/ml sigma Aldrich. It was kept for 16 hours at 37 degree Celsius and 5 % CO2. The oocytes were stripped by treating with 0.3% hyaluronidase.

IVF and embryo transfer

The motile sperm were prepared from sperm suspension that was gathered from cauda epididymis of CD 1 male mice by using a percoll gradient centrifugation. The PGC sperm was capacitated in IVF augmented with 3 mg/ml BSA, 5.46 mM for 30 minutes. Now, 15- 20 metaphase 2 oocytes were placed in 50 uL. That contain 1*10 sperm/ml and raise in mineral oil for 7- 8 hours at 37 degree Celsius and 5 % co2. Then, oocytes was washed 3 times in KSOM to erase sperm. After it fertilized oocytes was determined through 2 pronuclei. The CDI was gathered of mice with 5 IU of eCG for 48 hours and 5 IU of HCG for 14 hours. the 2PN zygotes were transferred to ovducts of 8 -10 week CDI female rat.

Histology and theca cell staining

The follicles which was cultured for 8 days was removed from bead and fixed for 2 hours in 4 degree Celsius in 4% in paraformaldehyde in 1* PBS. In ascending order follicles was dehydrated. The section were cut of serial 4 um and marked with hematoxylin and eosin. They were isolate in 3B hydroxysteroid dehydrogenase solution contain 0.12 mg/ml nitorblue tetrazolium chloride, 0.25 mg/ml beta nicotinamide adenine dinucleotide hydrate and 0.025 mg/ml epiandrosterone in 1* PBS for 30 min in room temperature.

Hormone assays

The androstenedione, 17 B estradiol and progesterone was measured of follicle in 2, 4, 6 and 8 days with help of radio immunoassay kits.

Statistical analysis

Here, one way anova analysis was used for measuring follicle size and steroid hormone. Similarly, oocyte size, IVF rate and maturation rate were also analyzed with 1 ways anova analysis with T test. Along with it, all statistical data was gathered by using GraphPad prism software.

Result

It is interpreted that oocytes in immature mouse follicles in alginate hydrogel were able to grow and result in birth of offspring. Hydrogel follicles had survival rate of 93.3% on 8 day culture and the average diameter increased from 156.1 to 348.8 in 8 days. Also, embedded follicles intact theca cell later and HSD staining. Moreover, steroid level increased in culture on day 4. The mean level arises from 1.27 to 2.12 ng\pl n some days. In that average progesterone was under1 ng\pl that increased to 2 ng\pl due to luteinization of granulosa cells. It is summarized that estradiol production increased to mean 0.19 to 4.29 ng\pl within 8 days.

Also, there was increase in oocyte from 61.78 to 68. 57 Um in 8 days. Similarly, the diameter grown in vitro ad vivo control was not same. thus, there is no significant difference identified. The mean of denuded oocytes of culture system is 82.3 %. Furthermore, the maturation rate of cultured oocytes was lower than that of control oocytes that developed in vivo, with a mean of 96.7% undergoing germinal vesicle breakdown and 91.9% reaching metaphase II

In IVF and embryo development the mean of 2Pn zygotes in fertilization was 69.2 % in vitro control and 81.7% in vivo control. Also, 20 2Pn zygotes was resulted from vitro control and 16 from vivo. Other than this, 2 female and 2 male pups were resulted from vitro control and 4 white pups from vivo control oocytes. Thus, all 4 mice were successfully fertilized in normal way.

The encapsulation of follicles been optimized to 93% survival rate and success of oocyte maturation released from scaffold was 70%. Also, it is analyzed that, 20% of live birth rate of embryos is improved in rate of 5.7% and 7.4% as compared to another report. There was no significant pre mature luteinization occurred between oocyte and somatic cells. The connexin 43 was demonstrated at same level. In granulosa cell, estradiol production increased after 4 days of culture. in addition, oocytes fertilized and implantation of embryos from vitro led to live birth.

Article 4 Monica et.al., Initiation of puberty in mice following decellularized ovary transplant

Methods and material

Human ovarian tissue acquisition and transport

It was collected from respondents who were undergoing ovarian tissue removal. In this 80 % of tissue was cryopreserved and 20 % were taken for research. The age of sample were 6 to 34 years old and were diagnosed from cancer. After that, tissue was sent to laboratory and kept at 4 degree Celsius for 14- 24 hours. from that a part was fixed in 10 % neutral buffered formalin. However, remaining was kept at room temperature.

Bovine ovarian tissue acquisition and decellularization

the bovine ovaries were gathered of young cows from Aurora packing company. They were rinsed in medium and excess fat was removed. They were cut into small pieces. The tissue was processed in 500 Um section of cortex and medulla in tissue slicer. Then, pieces were stained in solution SDS solution of 0.1 % and kept in room temperature. The similar process was done with human tissues. It is analyzed that piece of 5- 10 mm was removed, washed in deionized water and then stored in -80 degree Celsius. Each sample was sonicated in 2 ml tubes that contain 1 ml of 0.02% of triton X solution in ultra pure water for 45 minutes. Besides that, 1 ml liquid lysates were gathered and stored similar way. DNA of doubled stranded content was quantified using quanti - iT picogreen DNA kit.

Tissue fixation and histological analysis

Here, native and decellularized tissue were kept overnight at 37 degree. The serial section was cut 5 um thick and then slide of strain using Leica microsystems. The immunohisto chemistry process done on 3- 5 sections of sample. The experiment was done at least 2 times. After that pictures were taken. For visualizing DAPI counter strain with anti bodies were used in context of mouse histology.

Scanning electro microscopy

The process was performed by rinsing sample in deionized water to remove residues. Then, it was stored at -80 degree Celsius over night for removing moisture. Samples were then cut and mounted with carbon tape and coated with 15 nm osmium meta. Those who contained cells were fixed for 25 minutes in 2% glutaraldehyde, 3 % sucrose solution. However, SEM was prepared with 70 % ethanol and storing it in 4 degree Celsius. The cellularized sample was dehydrated in ethanol wash of 80, 90 and 100 % for 15 minutes. At last all sample was imaged using LEO 15 SEM at 3 kilovolt voltage.

Isolation of primary ovarian cells and seeding on bovine scaffold

The primary ovarian cell of 3- 4 week old mice was collected. The cells were cultured in plating medium of DMEM: F12 supplement with 1X insulin, 12. 75% nM HEPES , 1X penicillin, 10% FBS and 40 uG/ml hydrocortisone in 37 degree Celsius and 5% CO2. Moreover, dead cell, debris, RBC's etc. were washed in over night with PBS.

The scaffolds were generated from decellularize medulla section of bovine of 0.5 mm thick and 3 mm in diameter. Then, in sterilization of ethanol was used and PBS washing was done before seeding. On previous day, ovarian cells were collected through 0.025% trypsin EDTA and few cells were seeded in each scaffold in2 4 well plate with 400 uL of medium. The cells were cultured for 2 days at 37 degree Celsius with 5 % CO2.

Functional analysis of ovarian grafts

The surgery was performed 2 times. On 18 day CD1 mice was ovariectomized and anesthetized with ketamine. An incision was made in skin and in ovaries was done through subcutaneous layer. Then, fat part was separated from ovary and removed through top of uterine hor. After that, each incision was suture with non absorb suture. the incision was closed with 1-2 staples by applying lidocaine ointment. After surgery, ovary of mice draft was reopened and 3 mm graft was applied in kidney. Then, a 4 mm incision was made in capsule and kidney parenchyma was made with help of glass rod. Grafts was inserted into pocket under capsule. Similar process was applied with another kidney. After surgery renal graft was performed in 3- 4 weeks. The serum estradiol and inhibin were tested using GraphsPads software.

Result

Spalt like transcription factor 4 was expressed in metastatic leukemia cells and not in ovarian tissue. From out of 4, 3 contained SALLA 4 positive cells. The tissue sample contained primordial follicles and cell density. Also, tissue contained primordial follicles of cell density. The decellularized tissue was free of SALL4 antigen staining. Also, there was no visible nuclear material found in decellularized material.

In ovaries incubated in 0.1% SDS for 3 weeks at 4 degree Celsius, the cavities were clearly observed with eye. The SEM survey showed regions occupied by follicles, stromal cells and blood vessels. Furthermore, a primordial follicle is visible of 33.6 Um. Besides that, large follicle is visible in decellularized medulla. Moreover, lumens from spiral arteries are plain in it as well. It has been stated that structure of human ovarian tissue was observed as well via SEM. The tissue contained collagen bundles same as in bovine cortex. But the bundles were thick and uniform than tissue. While in immunohisto chemistry laminin is present in cortex. However, fibronectin is included in decellularized medulla and not in cortex.

In immuniohisto chemistry, out of 5, 4 ovary cortical tissue stained positive in collagen 1and 5 tissues contained collagen 4. But no decellularized cortex sample stained for laminin. Only 1 tissue stained positive for fibronectin.

The cellular preparation contained most ovarian cell types whereas RBC, tissue remnants and non follicles, oocytes was washed whereas granulosa and theca cells remained. The primary ovarian formed a confluent sheet on scaffold surface but some cell migrate through decellularized medulla within 2 days. Some granulosa cell organized into follicle pattern on scaffolds. The ovarian cells produce steroid blebs seeded on decellularized scaffolds.

The vaginal orifices verified closed in ovariectomized animal to xenograft surgery. The serum estradiol level of mice ranged from 5.8 - 10.6 pg/ml. whereas ovariectomized mice was from 5.1 - 6.7 pg/ml. it is analyzed from T test that there is significant difference in serum estradiol level of ovariectomized animal with graft as compared to sham group.

For evaluating health of mice grafts was examined. So, there was no gross rejection of graft on kidney. Both sham and recellularized graft contain immune cells. Also, granulosa cell formed follicle structure of 2- 5 cell. Moreover, alpha inibin and CYP17 cells were presented in grafts. The primary ovarian cell seeded in scaffold provided a niche for it. In one graft 2 antral follicle was included with exceeding 1 600 Um.

2. Comparison in difference of material, method and result between article 1 and 2

By analysing the articles, it is identified that there exist different between methods used and results obtained in article 1 and 2. The comparison is mentioned below :-

It is identified that in article 1 material used was female mice of age between 10- 20 weeks. So, the ovary of mice was taken and stored in -20 degree. Also, decellularization process was used that contained 0.5% of sodium dodecyl sulphate. Ovaries were kept for 16 hours in that and then washed by Dnase 1 solution for 30 minutes. The ovary was put in formaldehyde for 1 hr. Sections of 5 Um were cut. Also, immuohisto chemistry process was done. In order to label cells red fluorescent was applied. Here, quantitative calculation was done by using kits. Thus, ECM analysis was done on 6 ovaries. The scaffold toxicity test was applied on tissues. In addition to it, statistical test was applied using Shapiro Wilk test. So, qualitative data and information was included in it as well.

But on other hand, in article 2 there was no such ovary and tissue used to obtain outcomes. in this only secondary data was gathered by using articles, journals, etc. furthermore, the method used was to analyse articles to collect relevant and precise data. for this medline database was selected to chose articles from it. Besides that, in order to gather qualitative data secondary method was used. Thus, it clearly shows difference between material and method used. However, in article 1 primary data is collected by doing experiment and tests whereas in article 2 secondary data is gathered without doing any experiment.

In result it is interpret that in article 1 there was no difference between three recellularized ovarian scaffold types. But it was observed that there was large group variance. Alongside, it is observed that scaffold surface included large number of cells that confirmed ummunocyto chemistry and electron microscopy. There was presence of fluorescence in ovarian tissue as it is clearly scene in images of ECM structure. While in article 2 in vitro study follicles grow to a soft part and in vivo large follicles are lost in initial first day of avascular transplantation due to hypoxia.

3. Comparison in difference of material, method and result between article 1 and 3

It has been evaluated that there are certain differences in material and methods I article 1 and 3. They are defined as:

It is identified that in article 1 material used was female mice of age between 10- 20 weeks. So, the ovary of mice was taken and stored in -20 degree. Also, decellularization process was used that contained 0.5% of sodium dodecyl sulphate. Ovaries were kept for 16 hours in that and then washed by Dnase 1 solution for 30 minutes. The ovary was put in formaldehyde for 1 hr. Sections of 5 Um were cut. Also, immuohisto chemistry process was done. In order to label cells red fluorescent was applied. Here, quantitative calculation was done by using kits. Thus, ECM analysis was done on 6 ovaries. The scaffold toxicity test was applied on tissues. In addition to it, statistical test was applied using Shapiro Wilk test. So, qualitative data and information was included in it as well.

On contrary in article 3 animal used was 16 day old female and sperm of male mice of 8 -10 days old was taken. In this many methods were applied on different materials. Here, alginate hydrogel was prepared using sodium alginate containing 55- 65% guluronic acid. It was dissolved into water. However, encapsulation process was followed. In these ovaries was incubated in MEM. For measuring picture of encapsulated follicles microscope was used. The diameters were measured on 0 and 8 day. Along with it, oocyte maturation was done were oocytes was covered with 0.3 % hyaluronidase. Also, IVF technique was used to transfer embryo of mice. The PGC sperm was capacitated in IVF.

The theca cell staining was done to remove culture from bead. The section was cut into 4 Um and marked with haematoxylin eosin. However, radio immunoassay kits were used to measure follicle in specific day time period. The statistical test applied in this was anova along with t test and data was collected via GraphPad prism software.

In result it is interpret that in article 1 there was no difference between three recellularized ovarian scaffold types. But it was observed that there was large group variance. There was presence of fluorescence in ovarian tissue as it is clearly scene in images of ECM structure. While comparing outcomes it is said that in article 3 oocytes in immature mouse follicles in alginate hydrogel were able to grow and result in birth of offspring. The mean level arises from 1.27 to 2.12 ng\pl n some days. It is summarized that estradiol production increased to mean 0.19 to 4.29 ng\pl within 8 days. Furthermore, the maturation rate of cultured oocytes was lower than that of control oocytes that developed in vivo control analysis. 

4. Comparison in difference of material, method and result between article 1 and 4

By interpreting articles, it is identified the difference between material and method between article 1 and 4 that is explained below :

It is identified that in article 1 material used was female mice of age between 10- 20 weeks. So, the ovary of mice was taken and stored in -20 degree. Also, decellularization process was used that contained 0.5% of sodium dodecyl sulphate. Ovaries were kept for 16 hours in that and then washed by Dnase 1 solution for 30 minutes. The ovary was put in formaldehyde for 1 hr. Sections of 5 Um were cut. Also, immuohisto chemistry process was done. In order to label cells red fluorescent was applied. Here, quantitative calculation was done by using kits. Thus, ECM analysis was done on 6 ovaries. The scaffold toxicity test was applied on tissues. In addition to it, statistical test was applied using Shapiro Wilk test. So, qualitative data and information was included in it as well.

But in article 4 the material taken was human ovary tissue in which only 20% was taken for research. The material taken was of age 6- 34 who are been diagnosed from cancer. Also, bovine ovary of young cows was taken as material. It was then cut into pieces of 500 um section. Each sample was sonicated in 2 ml tubes that contain 1 ml of 0.02% of triton X solution in ultra pure water for 45 minutes. Besides that, 1 ml liquid lysates were gathered and stored similar way. DNA of doubled stranded content was quantified using quanti - iT picogreen DNA kit. The immunohisto chemistry process was applied on 3- 5 section. Also, elector microscopy scanning was done to remove residues. the SEM solution was prepared with 70% ethanol. The image of sample was taken using LEO 15 SEM at 3 Kilovolt voltage.

Apart from it, ovary cell of 3- 4 week mice is taken. In that surgery was performed 2 times. The fat was removed from ovary. After that ovary was opened and 3 mm graft was applied in kidney. then grafts were inserted into capsule and serum estradiol and inhibin was tested using GraphPad prism software.

In result it is interpret that in article 1 there was no difference between three recellularized ovarian scaffold types. But it was observed that there was large group variance. There was presence of fluorescence in ovarian tissue as it is clearly scene in images of ECM structure. In article 4 granulosa cell formed follicle structure of 2- 5 cell. In large follicle is visible in decellularized medulla. Also, there was no visible nuclear material found in decellularized material.

5. What makes to study article 1 different from article 2

The article 1 focus on scaffold generation of decellularization protocol. However, study focus on scaffold in context of composition, biocompatibility and mesenchymal stem cell in vitro. Also, various process and experiment was done to critically evaluate scaffold generation in mouse ovaries. The decellularization process was applied. However, ovaries were kept in SDS protocol. In addition to it, immounohisto chemistry process was applied and for labelling cells protein was used. The leica microscope was used to visualize image. Besides that, quantitative statistics were done along with ECM analysis. also, scaffold toxicity test was applied. At last statistical tests were applied such as Shapiro Wilk test. Hence, median and range was calculated in 10 -90 % range. It entirely focusses on mice ovary for reproductive bioengineering. In this both decellularized and recellularized process is applied. It is analysed that there is no difference in recellularization efficiency in large intra group variation.

The article 2 focus on advantage of biopolymer for fertility restoration in cancer patients. So, here data was gathered from previous journals and articles. In this secondary data was taken and analysed. There was no experiment done and statistics collected. however, the study focused on follicles and fertility preservation. It shows main biological actors that play vital role in maintaining structure of isolated tissue. Alongside, fibrin is protein that helps reproducing tissue engineering so that in cancer patient fertility is restored. Thus, fibrin is scaffold material used to preserve fertility. Besides that, study shows pros and cons of use of fibrin in fertility preservation. Furthermore, the relationship between fibrin and female fertility preservation is determined. Along with it, in vitro study the 3- D culture methods are mentioned and described. It also shows challenges of follicles in 3- D culture making. Thus, in depth discussion is done on fibrin and follicles. In vivo studies the connection between grafted tissue and recipient is shown.

Thus, both studies differ to a great extent. In first one mice ovary is taken as material for reproductive bioengineering application whereas in second study human ovary of cancer patient is taken for experiment. So, there is huge difference in that. In article 1 red fluorescent protein is used to label stem cells and only in vitro control analysis while in article 2 there is no fluorescent used to label cells.

6. What makes to study article 1 different from article 3

The article 1 focus on scaffold generation of decellularization protocol. However, study focus on scaffold in context of composition, biocompatibility and mesenchymal stem cell in vitro. Also, various process and experiment was done to critically evaluate scaffold generation in mouse ovaries. The decellularization process was applied. However, ovaries were kept in SDS protocol. In addition to it, immounohisto chemistry process was applied and for labelling cells protein was used. The leica microscope was used to visualize image. Besides that, quantitative statistics were done along with ECM analysis. also, scaffold toxicity test was applied. At last statistical tests were applied such as Shapiro Wilk test. Hence, median and range was calculated in 10 -90 % range. It entirely focusses on mice ovary for reproductive bioengineering. In this both decellularized and recellularized process is applied. It is analysed that there is no difference in recellularization efficiency in large intra group variation.

The third article focus on tissue engineering principles of culture of immature mouse follicles. For that follicles are designed in alginate hydrogel. Here, both male and female off spring of mice is taken. in this material used is 16 day old female mice prepubertal and sperm of CD 1 breed male. Furthermore, secondary follicles were also included for study. microscope was used for taking pictures. Moreover, embryo was transferred through sperm of male mice. Here, statistical analysis was used to measure steroid hormone, follicle size, IVF rate, etc. it is analysed that there is no significant pre mature luteinization occurred between oocyte and somatic cells.

So, difference between two study is article 1 method used was decellularization protocols. Also, microscopy was used to view images and scaffold toxicity test was conducted. For labelling red fluorescent protein was used. The mean elastin content in ovary tissue is 60%. There is no difference between 3 recellularised ovary based on density of cells.

Moreover, the hydrogel follicles have survival rate of 93.3% and diameter increased from 156.1 to 348. 8 on day 8. Vitro follicle is used in treating fertility where bioengineering is used in preserving fertility in ovary cortex transplant.

7. What makes to study article 1 different from article 4

The article 1 focus on scaffold generation of decellularization protocol. However, study focus on scaffold in context of composition, biocompatibility and mesenchymal stem cell in vitro. Also, various process and experiment was done to critically evaluate scaffold generation in mouse ovaries. The decellularization process was applied. However, ovaries were kept in SDS protocol. In addition to it, immounohisto chemistry process was applied and for labelling cells protein was used. The leica microscope was used to visualize image. Besides that, quantitative statistics were done along with ECM analysis. also, scaffold toxicity test was applied. At last statistical tests were applied such as Shapiro Wilk test. Hence, median and range was calculated in 10 -90 % range. It entirely focusses on mice ovary for reproductive bioengineering. In this both decellularized and recellularized process is applied. It is analysed that there is no difference in recellularization efficiency in large intra group variation.

The article 4 emphasis on clinical strategies to preserve fertility in female cancer patient. In that primary ovary cells were seeded into scaffolds. Also, ovary of mice 3- 4 week old mice was taken. Here, SEM method was used to visualize image by accelerating voltage of 3 Kv. The result obtained was that ovary tissue in of 3 out of 4 contained clear SALL 4 positive cells and participant D diagnosed with cancer did not find any positive cell. In immuniohisto chemistry, out of 5, 4 ovary cortical tissue stained positive in collagen 1 and 5 tissues contained collagen 4. But no decellularized cortex sample stained for laminin. Only 1 tissue stained positive for fibronectin. For evaluating health of mice grafts was examined. So, there was no gross rejection of graft on kidney. Both sham and recellularized graft contain immune cells. Also, granulosa cell formed follicle structure of 2- 5 cell. Moreover, alpha inibin and CYP17 cells were presented in grafts. The primary ovarian cell seeded in scaffold provided a niche for it. In one graft 2 antral follicle was included with exceeding 1 600 Um. Get MBA Dissertation Topics now!

Therefore, the difference is that in first 1 decellularization protocol was used and immounohisto chemistry process applied for labelling cells. And in 2nd one clinical strategy are determined to preserve fertility. This was done using mice ovary and SEM method was used to evaluate outcomes.

8. What are weakness in article 1

It is identified that in article 1 there are only few images provided of test and experiment are provided which can be used to see results. Along with it, the statistical outcomes are not represented in tabular format which makes it difficult to understand and analyse values. Also, images are difficult to evaluate. So, scaffolds observed are not visible properly. Thus, to visualise cells in cross sectioned recellularized scaffold was difficult. In this the figures highlighted are pasted in appendix so it is complex to identify and understand which figure indicate what thing.

9 What are strong points in article 1

The article stated strong points are that decullarisation with staining method clearly showed stained nuclei in the ovary of scaffolds. Moreover, it also showed removal of DNA after decellularization. From the image it was clearly observed that ovarian tissue enables in visualization of ECM matrix. Also, toxicity test conducted supported in determining removing of toxic remnants within process. the image also clearly showed green colour through which it was easy to identify fluorescence. Besides that, cell density is presented through a table of P1 P2 and P3. However, all keywords are mentioned at starting in article so it is easy to find out what each abbreviation shows. Beside that, the methodology is clearly been described in depth with each method stated. The study examined these scaffolds further, specifically with regards to the extracellular composition, biocompatibility and their ability to support mesenchymal stem cells in vitro. The labelled cells were seen clearly as red protein was applied in it. moreover, article is segregated into section that makes things easy to proceed in step by step. The figures are highlighted that makes it simple to gain knowledge that which figure indicate what things. other than this, in each figure its description is written below. So data and info is retrieved from it. the user is able to read description that what picture depict or shows.

10. What is similar in article 2 and 1

By interpreting the articles, it is analysed that there are certain similarities between article 2 and 1. First is they are based on mouse ovary for future reproductivity. In article 1 the mouse ovaries are taken as material for future reproductivity bioengineering application. In this ovary of 10 -20 weeks mouse ovary is taken. So, it focuses on reproductive fertility process. Here, vitro and vivo method are applied to study tissue engineered follicles. Similarly, abbreviations are critically described to make understanding of thing and words. In both article keywords are mentioned which make easy to analyse article. the methodology taken is similar as it include decellularization and microscopy to visualize images. Moreover, methodology is explained in depth which makes it easy to understand it. the tissue was sterilised in solution to remove fat from it. In addition, microscope was used to visualize images.

11. What is similar in article 3 and 1

By evaluating both articles it is find out that as method used in it are similar. Here, vitro and vivo method are applied to study tissue engineered follicles. Furthermore, decellularization and recellualrisation of ovary tissue is done. In the study material taken is mice ovary. However, another similarly identified is use of statistical method in analysing data and info. This is done to determine relationship between two variables. In addition to it, images are derived to represent and find out impact on follicles. Also, statistical method was applied both in vitro and vivo control analysis. the keywords are mentioned related to biology to find out important words. The images are clicked and its description is stated properly. The mean values are described in numeric terms in effective way in article.

12. what is similar in article 4 and 1

In these two articles as well there are certain similarities such as material taken is mice ovary. also, it is identified that in transplant of ovary tissue in fertilisation. Moreover, scaffolds in decellularization and recellularization has been analysed. the SEM microscopy was done to visualize images. Here, keywords were mentioned at starting to get a clear understanding and view of its meaning. The ovarian scaffolds were kept for few days that is in article 1 it was 14 and in article 4 it was kept for 8 days. Apart from it, the ovary tissue was cut off and kept in room temperature in 37 degree Celsius for 24 hours. The SDS solution was used to sterilised and water to wash tissue. the keywords are mentioned related to biology to find out important words.

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