The overall goal of this surgical invention is to produce autologous skin micro-grafts ready to be used immediately in the same surgery for wound healing purposes. This method can help answer key questions in the regenerative medicine and skin tissue engineering field about things such as chronic wound healing, burns and post-traumatic ulcers. The main advantage of this technique is that it's very easy to use by doctor without expectisile technique, and it is as fast and affordable.
To begin, use a biopsy punch to collect skin samples from a patient. Then remove and discard the epithelial layer. Combine each tissue sample with 1 milliliter of saline solution to generate autologous micro-grafts.
To prepare biocomplexes for clinical application, transfer 1 milliliter of the micro-graft solution onto collagen sponges. Immediately apply the biocomplex onto the patient's injured area. To in vitro, test the biocomplex's viability.
Following three days of culture, pour 0.3%formalin directly onto the biocomplexes and fix for 10 minutes at room temperature. Use a microtome to slice sections 5 micrometers thick and mount directly on a glass slide. Then place sections into 15 to 20 milliliters of xylene for three minutes.
Next, immerse the slices in decreasing grades of ethanol for one hour each before placing in deionized water to deparaffinize and rehydrate the sections. Then use 1 to 2 milliliters of 1 gram per liter of hematoxylin for one to two minutes to stain the sections. And transfer to water to rinse off the stain.
Now with 1 to 2 milliliters of 1%Eosin Y and 70%ethanol and dilute it in water, stain the sections for four to five minutes. Then use running tap water to rinse the slides. Immerse the sections in increasing concentrations of ethanol for one hour each before a one-hour incubation in xylene.
Finally, apply a based mounting medium to the samples before adding a coverslip. Then observe under a light microscope at 100x magnification. Using a dermatome, take 0.6 millimeter, 1 millimeter or 0.2-millimeter thick papillary, dead or dermis samples, respectively, from the trunk areas of four 40 to 55-year-old multi-tissue donors following national rules on harvesting.
Put the samples into 0.9%NaCl and place on an orbital shaker for five minutes to gently rinse the tissue. With a 5-millimeter biopsy punch, create samples that are uniform in diameter from the skin tissue, dead and dermis, and weigh all tissue specimens. Next, insert eight, three or four uniform samples of skin tissue, dead or dermis, respectively, into the tissue disruptor, and add 1.5 milliliters of saline solution for the disaggregation.
Carry out disaggregation for all tissue samples as indicated in this table. Use a correspondent number of punch biopsies derived from intact tissue samples as controls. After mechanical disaggregation, aspirate the saline solution containing micro-grafts and place each sample separately into a single well of a 12-well plate.
Add 1 milliliter of RPMI-1640 medium supplemented with 10%FBS and antibiotics to each sample. To evaluate the cell viability, to each well add 1 milliliter of medium containing 0.5 milligrams per milliliter of MTT solution, and incubate at 37-degree Celsius and 5%carbon dioxide for three hours. Following the incubation, remove the MTT medium and replace with 1 milliliter of DMSO.
After incubating for 10 minutes, transfer each sample in DMSO to a cuvette and use a spectrophotometer to read the optical density at 570 nanometers. Calculate the cell viability as the ratio of absorbance at 570 nanometers and the weight and grams of tissue used before disaggregation. Next, once the saline solution has been aspirated from the skin tissue, dead and dermis samples from a single donor, place each sample separately in a well of a 12-well plate for a cell viability test or a culture flask for morphological analysis.
Add 1 milliliter of RPMI-1640 with 10%FBS and antibiotics to the 12-well plate or 5 milliliters to the culture flask and culture at 37-degree Celsius and 5%carbon dioxide for 24 hours or seven days, respectively. At 24 hours, use light microscopy to carry out morphological analysis by evaluating the presence of cell suspension. Repeat on day seven.
With FACS analysis, analysis the dermis samples from mesenchymal and hematopoietic cell markers, such as a CD146, CD34 and CD45. Add medium to the desegregated cells, then incubate at 37-degree Celsius for three days. And assess the sterility of the tissue by performing microbiological analysis as previously described.
As shown here, human autologous micro-grafts immediately loaded on collagen support were implanted in a leg lesion. And a complete re-epithelialization associated with tissue repair was observed after 30 days. Moreover, the clinical followup showed a good texture and softness of the damaged area after five months.
As listed in this table, the thresholds of eight, four, and three biopsies that can be processed in a single step were established, and the tissues were disaggregated at four different times in order to identify optimal conditions of disaggregation to maintain good cell viability. The mechanical disaggregation demonstrated in this video maintains a mean value of 30%cell viability in all skin samples, dead and dermis, when evaluted at different times with respect to intact tissue. This graft shows that after 24 hours, or seven days in culture, no substantial variation of cell viability in skin tissue samples was observed by the time of homogenization in culture compared to starting time.
However, a reduced viability of dead samples after 24 hours in culture compared to starting time was observed. But cell viability was restored after seven days in culture. Similar results were observed for dermis.
When mastered, this technique can be done in five minutes if it is performed properly. While attempting this procedure, it's important to remember to remove the epithelial layer from the skin. Following this procedure, other methods like bone tissue disaggregation can be performed in order to answer other questions like bone healing mechanism.
After its development, this technique paved the way for researchers in the field of regenerative medicine and tissue engineering to explore the tissue healing in patients. After watching this video, you should have a good understanding of how to perform this micro-graft procedure. Don't forget that working with the tissue can be extremely dangerous.
And the precautions such as safety glasses should always be taken while performing this procedure.
