Epithelial Cells

Under general anesthesia, after performing the antisepsis of the sternum surrounding area, the puncture is done using disposable small diameter needles. This is performed in several places till an amount near 20 ce is obtained (Fig. 215-22).

The material obtained is centrifuged to obtain a high concentration of stem cells.

Stem Cells can be separated by many different techniques :

1. Directly: the material id centrifuged at low revolutions for a long time. The stem cells are then taken from the Buffy coat (the sample is taken including the neocytes layer) (Fig. 215-23).
Buffy coat is the area that contains stem cells, platelets and white cells (Fig. 215-23). .

2. In a Ficoll hypaque70 71 72 environment: stem cells are separated from unicellular by density gradient. This is a more effective separation method (there are no erythrocytes in the sample), but it needs one or two centrifugations more, to eliminate the Ficoll from the obtained cells because of its toxicity. This causes mechanical cell trauma because of the centrifugation.

For our objectives, we normally choose the first method because of its simplicity, not only because the final sample is not contaminated, but because it can be performed during the surgical procedure, at no risk of contaminating the sample.

Two smears are done to know the proportion of obtained cells and their integrity, one f them comes from the initial result of the puncture and the other one from the concentrated product.

Once the Stem cells are obtained, they are diluted in growth factor (normally a 1/20 dissolution is used). This dissolution is done for two reasons: to make the sample more fluid and to stimulate Stem Cells so their effect will be grater once applied to the area.

During this last period, we have been using stem cells that be obtain from bone marrow, to cooperate with surgical wound recovery, fat graft and hair implants and survival.

We base our choice on these criteria:

* Endothelial progenitor cells that stimulate vasularization, are located in bone marrow.

* Progenitor cells answer specifically to local trauma and ischemia.

* transferring all bone marrow’s stem cells, because of their plasticity, allows us to improve tissue recovery: skin, hair follicles, micro vasculature of removed flaps .

Conclusions
Our interest in following technological advances closely, takes us to using this new procedure, using growth factors and stem cells combined with fat grafts. We want to give plastic surgery a new cellular therapy to delay the aging process. There is still a lot to be discovered and understood n our path to eternal youth .

Fat Graft
Lipo-filling, lipoinjection, lipo-transference

Historical Summery
With the beginning of liposuction, fatty tissue became more important in the different surgical specialties since it can be injected to fill furrows or depressions.

The first paper about fat grafts was published in 1889 by Van der Meulen, He performed an epiploon graft between the diaphragm and the liver. In 1893, Neuber, presented a patient with infra orbital depression because of TBC treated with multiple and small grafts obtained from the arm.

The first report of fat graft used as a mammary implant to increase or reconstruct the shape was done in 1895 by Czesar. Later, in 1909 Lexer reported a case of hemifacial atrophy that he corrected using a fat graft block (4.72 x 4.72 inches). He also discussed certain conditions that are necessary for the procedure to succeed. One of them is that the receiving area had to be prepared carefully and that the transplant had to be done immediately.

Kanavel, in 1916, suggested that graft survival improves if it is not attached by sutures. He emphasized the importance of asepsis and hemostasis to obtain better results. But many grafts in all the studies done till that date showed some sort of reabsorption of the fatty tissue that was transplanted. This is how in 1919 Lexer again, advised about over-correcting because of 45% reabsorption of the transplanted material after 14 months. He suggested using large sized fat grafts, since he postulated that smaller ones will suffer from fibrosis when the larger ones would remain as fatty tissue.

Same author presented in 1913 a cystic mastitis case in which all the suffering tissue was removed. The defect was later filled. He concluded that irrigation to nourish the graft was insufficient causing its complete reabsorption.

During the first four days after the procedure, there is an extensive cellular infiltration of lymphocytes, polymorphonuclear cells, plasmatic cells and eosinophils from the host. In the graft´s blood vessels, red cells remain accumulated while white cells start diapedesis. Endothelial cells, fibroblasts and fat cells have few changes. On the fourth day even though blood vessel remain collapsed their stromal cells thicken, red cells increase their number. This means that there was a vascular anastomosis between the small vessels from the graft and the host´s vessels. The infiltration now has an increased number of eosinophiles and giant cells. These cells will initiate the degeneration process that becomes more evident on the fourteenth and twentieth day. Fat Cellular Digestion takes place and the histiocytes phagocyte the lipidic accumulations saving them in blisters. By the end of the month these cells seem to have finished their job and finally the fat cells is reabsorbed and replaced by fibrosed tissue.

These two observations were the base to develop two very important theories:

1. Cellular replacement theory

2. Cellular survival

The first one suggests that histiocytes replace lipocytes; the second one, that histiocytes attack only some lipocytes and phagocyte the lipids instead of replacing them, this way some cells would survive the immune response.

It was Hausberger that presented the prelipocytes theory, which postulates that these cells are destined to be mature lipocytes cells. A two year investigation performed in both mice and humans proved that there is no cellular replacement, but transplanted adipocytes are revasculariced in a very small volume. In 1945, he performed an experiment where he obtained immature fatty cells and five days later he replaced them to the same rat. These cells´ developments begin seven days after been transplanted.

In 1956 Peer enunciated hi “Cellular Survival theory”, which depended on early vascular neo-anastomosis and on the dermis´ availability to facilitate nutrition.

In the 80´s, microscopic studies of cellular behaviour were the latest trends, and after different analysis and with the possibility to reproduce results, fatty tissue was recognised as an organ.

In Argentina, the biggest advance was Dr José Viñas´ introduction to the importance of graft preservation as a concept (published in 1973). In Brazil, Raul Loeb published in 1981 the auto-fat graft to palpebral bags, in secondary eyelid surgery depressions and in senile looking orbits.

Many articles were presented about fatty tissue applications as a treatment for unaesthetic deformation. Some of them were done by Teimourian, Illouz and Chajchir.

In 1982 Dr Illouz presented the first experiences of fat obtaining by liposuction to be re-injected in depressing areas as filler.

In 1984 Ellenbogen, reported preliminary results about using fat raft pearls. The pearls´ diameter was not larger than 6mm. he used them to fill trauma defects, acne consequences, naso-labial folds, eyelid defects and chin augmentation.

There are studies that show that fat-grafts do survive while other studies establish that the correction happens because of a fibroblastic and inflammatory local reaction; and some other studies, that the fat´s remaining is very short.

In 1990, we started an investigation using laboratory animals, about transplanted fatty tissue´s reabsorption and survival. The results were published in 1993.

On the other hand, in Mexico, professor Guerreosantos, established an important relation between the vascular penetration index to the fat-graft and its survival capacity through an experimental study done in 120 rats. He proved that blood vessels penetrate fat-grafts. Fat-grafts are also completely permeable to the new blood vessels provided by skeletal muscle and fascias.

This study included a 12 month follow up, but it did not prove a noticeable volume reduction. The same author published in 1996 his 12 year experience in using autologous fat-graft as a facial and body shape filler with good and permanent results.

Today, fat-grafts are used in many areas like

Orthotics,

Traumatology: joint anchylosis and osteomyelitis;

Neurosurgery: skull and dura mater defects, nerve protection, laminotomy ;

Thoracic surgery: defects on the thorax wall and pleura.

Ophthalmology: to repair orbital cavities

Otorhinolaryngology: auricle reconstruction, frontal sinusitis, mastoid surgery, mouth-nose communications, depressed palate Perivaginal injuries, Prostatectomy defects .

Patient Selection

Pre-surgical evaluation
Each case needs to be studied according to the highest clinical standards and the area that will be treated will have an operational plan designed according its defect. The patient will undergo several laboratory analysis and a pre-surgical check up which is a routine in surgical interventions. A photographical study is essential in order to be able to do a follow up of he case.

Depending on the donor area and the receiving area general anesthesia, local or local combined with sedation might be indicated.

It is crucial to provide a clear explanation about the method that is being used and long term result so the patient will know what to expect .

Technique
There are many ways to process fatty tissue after it was obtained by liposuction. One of the most popular techniques is centrifuge to separate the lipocytes from free lipids and serohemorrhagic liquid.

Another procedure is to wash the fat with a 5% glucose solution or with normal salt solution to obtain blood free fatty tissue or to add other substances to increase adipocytes survival.

Finally, sedimentation can be used to separate adipocytes by decantation; a cloth can be used in this case with out modifying adipocytes´ structure.

We study adipocytes survival with different techniques to process fatty tissue. This study established that adding insulin or other substances does not cooperate to fat cell´s survival, and that centrifuging at a high or low speed produces cellular destruction, making its survival less viable.

After more than twenty years of experience, following patients and having tested the different methods we mention here, we think that best results are obtained be sedimentation with no additives. We also see that long term results show this procedure´s success.

Because work with autologous material, results last longer, no rejection process is activated, the material is easy to obtain and the procedure could be done several times.

This procedure offers great advantages when compared to synthetic implant .

Our procedure

Once the asepsis and anesthesia are done, we perform liposuction in a dry way when possible, that is without infiltrations, if the graft is small.

When the area is large, we perform a tumescent technique (Klein solution is infiltrated) to reduce the bleeding (Fig 215-24 and 215-25)

Fat removal is done with 6 to 8 mm blunt nosed cannulas to avoid the destruction of the cells (Fig 215-26). The cannula is connected by a sterile polyethylene silicone tube to a trap flask. This flask is connected to a vacuum pump that has a 10 mercury inches per 2 cm vacuum (Fig 215-27).

This vacuum has been specially calculated in previous studies not to harm the cells, because at a higher pressure, these cells would collapse (vacuum chamber experience).

In minor procedures, when the fat sample is obtained with a syringe, the piston can not be at the top, but just a third for the aspiration to be done correctly. We recommend a negative pressure of half an atmosphere to obtain the tissue as healthy as possible. We collect it in a sterile recipient and we pour it in a syringe, taking special care on the fact that fat´s colour needs to be yellow and the sample, oil and blood free.

When the material is fluid free, it can then be injected directly. N the case the sample had salt solution, water or blood, it is recommended to decant the sample. Once this is done, the fat will be placed in syringes to be injected to the receiving area.

The sample should not be washed, nor filtrated nor mix with additives or anesthetic solutions, because like we said before, these external factors modify the graft´s attachment and survival .

We have performed histopatholigical studies f the fatty tissue removed by liposuction when hypodermic adipocytes can bee seen in lobule.

In some areas, they might be arranged around connective tissue: inter lobular septum (Fig 215-28)

Most adipocytes show a preserved structure with peripheral nucleus against the cellular membrane. Between the cells normal capillaries can also be appreciated. There might be some destroyed areas and different sizes vacuoles formed by the accumulation of adipocytes. Some red cells can be seen because of recent extravasation .

Fig 215-28.A. H-E Staining. Adipose Lobules separated by connective tissue.

Fig. 215-28. B. PAS –E Staining. Connective tissue and some peripheral capillaries.



Fig. 215-29. A and B.

Other samples were taken three months after the surgery avoiding areas of cytoesteatonecrosis, granulomas, lymphocytes, adipocytes, multy nucleus cells and great number of neo formatted vessels.

Six months later, the histological sample shows a “Swiss cheese” pattern and poly nucleus cells, that match the granulomas. There is also a lymphocyte reaction and peripheral fibrosis.

One year after the procedure, biopsies showed more corrective tissue and fibrosis. Other authors have done histopatholigical studies comparing the material processed with the different techniques of autologous fat-graft, observing that the sedimentation method preserves a greater amount of visible adipocytes that centrifuge and washing method.

Fatty tissue is injected with 1mm syringe and with half of a mm blunt nosed lateral ending cannula, depending on the area that´s been treated. Syringe with smaller diameter would cause great destruction because of their pressure.

Injection is done as retro-injection, this means that it injects when one removes the needle, leaving remaining material in the graft area. (Fig 215-29 and 215-30). The indicated place to do so is under subcutaneous tissue, near the fascia and muscle, to reassure a better irrigation and a better attachment. The needle is introduced following a previous mark (Fig 215-31) .

Fig. 215-30. The cannula is introduced and deposits fat in cylinders by retro-injection.

Fig. 215-31. A. Incorrect technique. B.
Fat cylinders with the proper muscular and subcutaneous distribution.