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The use of confocal microscopy establishes methodology to count the keratocytes in the corneal stroma, and measure the density of the ADAScs in the implanted tissue, as well as in the corneal stroma.
This article was reviewed by Monal El Zarif, OD, Msc
New evidence with confocal microscopy shows that adipose-derived adult stem cells (ADAScs) injected into the stromal pocket of patients with keratoconus can transform into keratocytes, according to Mona El Zarif, OD, Msc.
The use of confocal microscopy to monitor and evaluate the evolution of the stem cells over time is novel, said Dr. El Zarif, a doctoral student at Miguel HernÃ¡ndez University in Alicante, Spain, at the European Society of Cataract and Refractive Surgeons (ESCRS) annual meeting.
“It allowed a qualitative and quantitative assessment of the cells,” she explained.
The technique establishes a new methodology to count the keratocytes in the corneal stroma, and measure the density of the ADAScs in the implanted tissue, as well in the corneal stroma she said.
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The researchers compared three groups of adult patients with keratoconus in an experimental, interventional, prospective, consecutive case series. In the first group, five patients were implanted with ADAScs alone without scaffold.
In the second group, five patients were implanted with decellularized human corneal stroma, essentially a scaffold without ADAScs.
“We decellularize the lamina from the donor human cornea stroma,” Dr. El Zarif noted.
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The third group was implanted with ADAScs embedded onto a scaffold made from a decellularized lamina.
“We observed that the automatic count of cells done by the software of the confocal microscopy was not accurate,” said Dr. El Zarif. “That’s why we proceeded with a manual count of cells. We proceeded choosing a good image, a good number of keratocytes and good contrast and quality of illumination.”
For anyone wanting to try this approach, she recommends starting the count of cells with 50% illumination and contrast determining a fixed area.
In this study, Dr. El Zarif analyzed an area of about 0.1 mm and counted the most illuminated and most refringent cells with well-defined edges.
“The dark gray cells are not counted in our study because they don’t belong to the plan of observation,” she said.
When the researchers could not find any defined structure similar to keratocytes, they considered the tissue in question to be acellular.
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By this definition, the decellularized lamina in this study remained acellular a month after implantation.
By contrast, cells in the recellularized lamina after one month had structures similar to normal keratocytes Fand these cells become more similar to keratocytes six to 12 months postoperation.
The researchers used software in the confocal microscope to calculate the density of the cells. They defined cell density as the number of cells multiplied by 10 (cell/mm2) +/- SD.
The results were promising. In the first group, those implanted with ADAScs but no scaffold, the ADASc appeared round in shape, and more luminous, refringent, and voluminous. The shape of ADASc changed after 6 months from round to fusiform until after 12 months the morphology of these cells looked like a normal cornea.
In the second group, those patients implanted with decellularized lamina only, the lamina remained acellular for the first month after implantation.
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After three months, it became recellularized by the patients’ own keratocytes.
“There was a migration of keratocytes from the host stroma toward the lamina,” said Dr. Zarif. The posterior surface of the lamina recellularized before the anterior surface. After a year, the keratocytes populated the lamina in a similar way to a normal cornea stroma.
The third group, implanted with recellularized lamina, showed structures like normal keratocytes after one month. These were more voluminous in the posterior surface of the lamina. With some patients they had dendritic shape after one year of implantation.
In all three groups, 12 months after the surgery the researcher noted gradual and significant increase in the cellularity of the anterior and posterior stromas of the patients in comparison to the preoperative density, with a p value for this difference of less than 0.001 in the anterior and posterior stroma.
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Mid corneal stroma
As the study progressed, the research team continued to make new discoveries in each of the groups.
In the first group, the researchers noted a gradual but significant increase of the cellularity of the mid corneal stroma (p < 0.001).
In groups 2 and 3, one year after surgery, the researchers observed a significant increase (p < 0.001) in cellularity in the anterior and posterior surfaces and within the lamina.
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They found that cell density was significantly higher in patients implanted with recellularized laminas than in those implanted with decellularized laminas, both on the anterior surface and within the lamina (p =0.011), and on the posterior surface of the lamina (p = 0.029).
After making its initial findings on cell density, the research team continued to follow up on this measurement.
After one year, group 3 had greater cell density than either group 2 or group 1 in the anterior stroma. It had greater cell density than group 2 in the anterior stroma, the anterior surface of the lamina, the mid stroma of the lamina and the posterior surface of the lamina.
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In conclusion, Dr. El Zarif said, confocal microscopy is an essential tool for the evaluation and monitoring “in vivo” of the ADASC.
“It allowed a qualitative and quantitative assessment of the cells through the experiment,” she said. “And it allowed monitoring of the progressive morphological changes that occurred in the implanted tissue in decellularized and recellularized laminas.”
Dr. El Zarif added that the technique assisted in determining the change in the cells’ densities in the grafted tissue as well as in all the posterior and anterior corneal stroma.
Mona El Zarif, OD, Msc
Dr. El Zarif has no financial disclosures. Read more by Laird Harrison