Best Techniques to have
a Successful Fat Grafting Outcome

Best Technique to have a
Successful Fat Grafting Outcome

Although fat grafting is generally safe with good patients’ satisfaction, many still experience unpredictable outcomes with varying fat cell survival as the adipocyte are less robust than stem cells & require a non traumatic method of harvesting to prevent cellular injury & subsequent apoptosis or phagocytosis. Depending on the technique and time that is required for harvesting &  lipofilling, 40–90% of the injected fat graft volume will remain, while the rest is resorbed within months after grafting. Oily cysts may remain in the grafted area as a consequence of this fat necrosis.

Best methodology of fat transfer, best technique that can gives us:

  1. Highest fat retention, which indicate high percentage of fat survival which can end good surgical result and least unsatisfactory outcome.
  2. Highest retention depends on highest fat cells survival plus highest survival of stem cells in the fat graft to play its major role of fat survival
  3. High quality fat graft depending on the  fat aspiration, processing & injecting techniques that are used and need to be considered carefully.

Every step-in fat transfer that consists of harvesting, processing & administration may have

“Deleterious effects on cell viability”

Process of Fat Transfer

1. Lipo-aspiration (Harvesting the Fat) Technique:

Simply, harvesting require two pieces of equipment:

  1. A suction source
  2. A collection receptacle

The lipoaspiration cannula can either be connected to a syringe, so that aspiration can be carried out manually or using liposuction machine to aspirate the fat & this mainly for large amount fat transfer procedures.

Whether the cannula connected to a syringe as the suction forces created by retracting the plunger of a syringe or by external suction machine, the key factor is not only by what we are suctioning but also, how much the negative pressure that we are creating during suctioning. 

About 10-15 % of cells were lost when the suction on or near maximum negative pressure ( -28 in. Hg (700mmHg) or one atmosphere) (Shiffman et al. 2001).

Small amount lipo-transfer:

 The surgical tool to aspirate fat is highly important from design & size of cannula to the negative pressure inside the syringe. Pulling of the plunger should be carried out with caution because it will determine the negative pressure inflicted on the tissue. The negative pressure caused by syringe is a critical factor in graft survival, few who they know that by syringe can cause negative pressure higher even than conventional liposuction machine, it could reach up to 700 mmHg. Whileany negative pressure above 500 the fat should not be used for lipotransfer. With this in mind, special syringes with a controlled plunger can be used.

A prospective, randomized, comparative study demonstrated that the main negative pressure of 10,20,60 ml syringes were 275, 394, & 549 mmHg, respectively, & NO significant differences in adipocytes & stem cells integrity & viability were observed (Charles-de-Sa et al.2015).

Pu el al. (2008), looked at the cellular function of adipose aspirates using syringe technique & conventional liposuction on the same patients, they found that a significant higher quantity of viable adipocytes in the Coleman technique ( Syringe Technique). 

A lipofilling study by (Witort et al.2007) evaluated the effects of different harvesting techniques on adipocytes, the results indicated that the gentle syringe technique (Coleman technique) was less traumatic than the mechanical aspirator (680 mmHg vacuum) which uses power-assisted aspiration (Witort et al. 2007). These results were supported by Herold et al. (2011) who compared the fat graft viability of adipocytes using the Coleman technique to the Shipper technique. The Coleman technique involves manual aspiration using as syringe & centrifugation, while the shipper technique uses automatic liposuction & no centrifugation of adipose tissue & found Coleman technique was superior with significantly increased fat graft viability (Herold et, 2011).

Large amount lipotransfer:

In large amount fat transfer (Mega Lipo-transfer) like fat grafting for buttock, breast or multiple areas; a liposuction machine has to be used to decrease the time of surgery. Most plastic surgeon world-wide currently in practice, with experience in different types of liposuction techniques.

  • Laser Lipolysis, LAL, produces nonviable adipocytes.
  • Suction-assisted liposuction SAL, being the preferable, but less viable adipocytes & stem cells compared to the Coleman harvesting technique (Myer et al. 2015).
  • The water-jet assisted device WAL(Body-Jet, HumanMed, Hamburg, Germany) technique, WAL, there are more intact cell & normal fat architecture & higher adipocytes & stem cells survival than ultrasonic liposuction machine.
  • Evaluation of energy-based systems, 3rd generation-ultrasonic-assisted liposuction (UAL) has been shown benefit of 53% better skin tightening & up to 30% less blood loss than standard liposuction. Histological examination of lipoaspirate revealed 85% adipocyte & 87% stem cell viability, respectfully, (the energy levels were V- mode with 60% amplitude or C- mode at 50% energy amplitude).

In conclusion: The body of evidence does not support one harvesting technique above another as superior. However, technique that use lower-pressure suction by means of large-bore cannulas appear to increase adipocyte viability. If a higher pressure is used during harvesting, then its best to employ fat-processing methodology to include filters, washing, & /or centrifugation to removed dead cells & oil. Some of fat collection containers contain filters, the pore size of a filter 500-800 µm (800 µm nominally better than the 500 µm filter size), this filter is very effective for removal of oil and extracellular fluid & retained viable adipocytes & stem cells. 

Factors Affecting the Outcome (How to get the best stem cell quality from adipose tissue)

  1. Donor Site

As far as fat viability & graft outcome are concerned, several studies have been unable to demonstrate a significant difference between harvesting sites. Recently it was confirmed that the most common source for the harvesting of fat grafts is the lower abdomen (abdominal wall is significantly higher than flanks areas).

  1. Cannula Size:

Ozsoy et al. (2006) demonstrated in his prospective study that a greater number of viable adipocytes with a 4-mm diameter cannula compared with the smaller 2 – or 3-mm cannulas, this also supported by Erdim et al. (2009) as they recommended the use of larger cannulas to increase cell viability.

  1. Fat collection containers:

Exposure of fatty tissue to open air in histological studies has demonstrated cell membrane lysis of up to 50% of cells exposed for a 15-minutes duration. Closed collection containers & syringes are now the standard of care because of the air exposure effect from cell desiccation, bacterial contamination, and cellular death.

  1. Time to Fat Grafting:

Adipose tissue cell viability from 1- 4 hours post-harvesting showed an increased loss of adipocytes over this time period. although the scanning electron microscopic assay indicated  No remarkable anatomical changes based on preservation methods, oil volume significantly increased in fat preserved at room temperature for 4 hours (Matsumoto et al.2007), they concluded that adipocytes should be transplanted as soon as possible if stored at room temperature. The processing should perform as soon as there is enough fat collected to process and then the fat, with or without concentrated stem cells, is immediately grafted into the planned recipient site. 

  1. Purification Step (Processing of Harvested Fat):

Aims to remove the interstitial liquid whilst at the same time ensuring the viability of the graft cells. When choosing a processing method for fat grafting, the volume retention, viability, & vascularity of the resulting graft are important aspects to consider. The three main widely used nowadays purification protocols are, namely,

  1. Decantation
  2. Centrifugation
  3. Filtration Method (Washing)

Decantation

The decantation process is easy to perform and does not necessarily require any additional devices. It can be performed directly in syringes or in a special device designed to trap the fat tissue and to isolate the liquid fraction, it is probably the cheapest & easiest to use for surgeons, the main limit of this process is that a significant fraction of liquid will still be trapped in the adipose fraction and will be the first to be reabsorbed after reinjection. The remaining infiltration liquid can also be responsible for a higher concentration of pro-inflammatory cytokines secreted by the adipose tissue during aspiration. This may trigger inflammation at the recipient site, with recruitment of immune cells that can eventually lead to increased graft resorption.

Centrifugation

Due to this limitation in the decantation process, since the 1990s the lipoaspirate can also be centrifuged to remove the liquid fraction and improve graft uptake.

Centrifugation of adipose tissue separates fat cells from lipid, blood cells, water, and water-soluble ingredients such as proteases and lipases, but does not shift ADSCs between the adipose and fluid portions, possibly due to the strong adherence to adipose tissue or since they are resident with the adipose tissue.

  • The short-term survival rate of aspirated adipose grafts per volumetric unit after centrifugation increased with centrifugal forces up to 3000 g-force.
  • Older fat cells are relatively larger in size than the younger fat cells. Squeezing the fat cells by centrifugation enables selective destruction of larger and older cells that tend to be more fragile.
  • Excessive centrifugation can destroy adipocytes & adipose-derived stem cells, but appropriate centrifugation concentrates them.
  • Centrifugation does result in increased cellular destruction; however, it’s from the older, larger, or more injured fat cells.
  • They found that 1200 g-force (3000 rpm) is the optimal centrifugal force for obtaining good short & long-term results in adipose transplantation. Yoshimura el al. (2006)

It is shown also that the increased centrifugal forces compacted the adipose portion more and therefore concentrated the red cell within the adipose portion rather than shifting the red blood cells into the fluid portion.

Butterwick et al. (2006) showed in their prospective, double blind, randomised study compared centrifuged with non-centrifuged fat grafted to hand that the centrifuged fat demonstrated a clear advantage in longevity and aesthetic outcome measured at 5 months post-treatment.

In contrast, Khater et al. (2009), found an improved clinical outcome with the washed at one year compared to centrifuged fat.

Botti et al. (2011), compared one side of with centrifuged fat (3000 rpm for 3 min.) with another half filtered & washed. At 12 months, there were NO subjective or objective differences in outcome.

Ferraro et al. (2011), their study suggesting that lowering-force of the centrifuged specimens improved cellular viability. They also showed less resorption in centrifuging compared to simple decanting alone.

Kurita et al. (2008), They found that 1200 g-force (3000 rpm) is the optimal centrifugal force, as it lead to condensation of cell numbers per volume of adipocytes & ADSCs with an improved ADSCs to adipocyte ratio despite the number of ADSCs contained in the adipose portion not changed by the centrifugation.

Likewise, Kim et al. (2009), evaluated the centrifugation technique on fat cell viability in autologous fat transplantation. They concluded that excessive centrifugation with 5000 rpm for more than 5 minutes increased adipocyte destruction and recommended that centrifuged force be limited to 3000 rpm (1200 g-force). Furthermore, when centrifugation is used, several of articles suggested that forces greater than 3000 rpm (about 1200 g-force) causes more cellular damage.

Removal of the lipoaspirate liquid component seems to be critical to obtain the best graft maintenance, at the same time centrifugation can be harmful to the tissue when the duration & speed are excessive.

The use of several short & soft centrifugations overcomes these constraints while at the same time concentrating the fat tissue.

The other advantage of using multiple centrifugations is that it allows washing steps to be included to eliminate possible dead cells, residual local anaesthetics, and cell debris.

In conclusion: The body of evidence does not support one processing technique above another as superior.

Filtration Method (Washing):

One of the simplest & yet most efficacious methods for fat processing is preparing the lipoaspirates by washing with normal saline. Washing steps are crucial to reduce undesirable components such as blood, debris, free lipid, & ruptured adipocytes to purify the fat before reinjection while retaining viable adipose tissue for further use. The most common release criteria value for cell viability is above 70%

According to Conde´-Green et al. (2010):

  1. It is the best processing method
  2. Maintains adipocyte membrane integrity
  3. Maintains adipocyte numbers
  4. Clear most blood contaminants
  5. Increases the number of ADSCs and endothelial cells

Moreover, Conde´-Green (2010), found a greater concentration of ADSCs in the centrifuged fat than with the decantation method alone. Caution must be taken with this centrifugation step to preserve the integrity of the adipose tissue.

High-speed centrifugation resulted in consistent volume retention but lower viability. Each of these approaches is ideal under different circumstances & contributes to the versatility and reliability of fat grafting.

Some protocols also integrate multiple washing steps (e.g., Macrofill by Adip’sculpt; PureGraft by Cytori) that aim to remove the remaining infiltration solution, to get rid of most of the local anaesthetic, vasoconstrictors, inflammatory molecules, and death factors (released during liposuction) that may damage the tissue and limit graft success.

It is accomplished by passing the lipoaspirate through a filter (e.g., PureGraft by Cytori) to remove particles below the chosen size. In 2013, Zhu found less oil and greater viability with this system compared to high-speed centrifugation.

Coleman, (2004) states that washing, although advocated by a number of authors, is not recommended, because in this experience the mechanical action of washing subjects the reticular fibres and connective tissue septa to unnecessary trauma and can disrupt the fragile fatty tissue architecture.

6. Fat Administration (Reinjection of Harvested Fat):

The final step of fat transfer is the reinjection of the adipose tissue. Inevitably, some degree of hypoxia occurs around the grafting of the lipoaspirate. In the recipient, the integration of the graft requires extensive (re)vascularization. Adipocytes are sensitive to hypoxia & therefore prone to death and necrosis. This step usually requires the use of different sized syringes (from 1 cc to 50 cc) depending on the quantity of adipose tissue to graft. Too large ‘lumps’ of fat transfer obviously develop necrotic cores due to diffusion insufficiency, as a result of which the graft ‘take’ may be reduced due to hypoxia  & fat cells death. Best methodology to avoid massive fat necrosis is require “Multi-levels Fat Grafting” in the recipient area by which, small amount of fat tissue injected by retrograde movement that deposes fat cells in multiple levels by micro-tunnelling and in multiple directions

Micro-Lipofilling
Multi-levels Fat Grafting
Micro-Lipofilling
Multi-levels Fat Grafting

Therefore, the injection of small aliquots of fat is now commonly accepted, care must be taken not to over-graft in the donor area as the amount to inject judge by the desired outcome plus tolerance of donor area.  Faced with resorption, many surgeons try to overcome this limit by ‘‘over-correction’’ of the recipient site. However, exceeding the volume might lead to an inverse effect with increased graft pressure resulting in tissue damage, necrosis of fat & more chances of oil cyst formation.

More Complex Protocols

Due to unprotectable fat resorption rate, methods to increase graft viability are needed. In the last five years, a new approach has been developed to improve fat grafting through the addition of stem cells or growth factors

Platelet Rich Plasma (PRP) Enriched lipotransfer:

In surgical fields, great interest has been demonstrated in platelet concentrates, notably in PRP, which is easy to use and only requires a blood sample. This blood derived formula contains a concentrated number of platelets that releases several growth factors. In Gentile et al. (2012) reported a 1-year graft survival of 69 % PRP-enriched compared to 39 % for the Coleman technique alone. PRP clearly has a concentration-dependent effect, so, more studies with a higher number of patients will be required to reach a consensus on the use of PRP at a defined concentration.

 Stem-Cell enriched Fat Transfer:

1. Cell-assisted Lipotransfer (CAL)

Over the past decade, studies have shown a great deal of interest in the SVF mainly because of its ASCs content and its ability to secrete pro-angiogenic factors. Thus, new procedures have emerged, consisting of the injection of a SVF- enriched fat graft. The common name for this protocol is Cell-Assisted Lipotransfer (CAL). The goal of this approach is to inject more SVF cells due to:

  • The multipotent capacity of stem cells in SVF allow them to differentiate into endothelial cells which promote vascularization and increase blood supply to the fat and survival of the graft plus secretion of growth factors by the SVF which can increase blood to fat.
  • The multipotent capacity also enables the stem cell to differentiate into adipocyte to overcome the death of adipocytes that occurs during and after fat transfer.
  • Finally, the immunoregulatory potential of ASCs may decrease the inflammation that is responsible for a sub-optimal outcome, causing adipocyte death.

In the CAL protocol, the adipose tissue is harvested by liposuction, half of the harvested fat is used to prepare SVF as the lipoaspirate is subsequently enzymatically digested by collagenase and fractionated in to SVF. the SVF then combined with the other half to create a stromal cell enriched fat graft. Several devices are now used in clinical practice to isolate the adipose SVF. These devices often use enzymatic digestion to digest the tissue & allow the isolation of the SVF cells as a pellet following centrifugation of the digested tissue.

In the past 10 years, many research groups have shown interest in Cell-enriched Lipotransfer (CAL) protocol (Fat with SVF),  SVF seems to improve scar healing and might be suitable for low vascularity recipient sites but still not clear if this protocol is better than conventional fat grafting. It might be promising for hostile & poorly vascularised tissue sites (an irradiated breast for example) but not sure if it is:

  • Justifying the higher cost
  • The necessity to harvest more tissue (half of it is digested) and the
  • Supplementary operation time that is required (approximately 2 h more).

The present evidence suggests that there is a big potential for CAL in reconstructive surgery as seventeen studies examined weight/volume retention of which 15 studies favoured CAL over conventional lipotransfer. One clinical study did not find any efficacy of CAL, however, the present studies are so far still of low quality with inherent weaknesses. Several aspects regarding CAL remain unknown such as the optimal degree of cell enrichment and also its safety.

1. ADSCs-enriched Fat Transfer (Cultured Stem Cells)

Since the study published by Trojahn Kølle et al. which carried out with volunteers in which fat grafts were compared with fat grafts enriched with cultured ASC. The result of study showing the ADSC-enriched fat grafts had significantly higher residual volumes (80.34 %) compared to non-enriched fat graft (16.3%) after measuring it by MRI. The difference between the group was scientifically very significant and no any serious adverse event were noted. This study has raised the interest in cultured adipose derived stem cell supplemented fat graft (ADSC-enriched fat grafting) as it had an excellent feasibility & safety.

These promising results add significantly to the prospect of stem cell use in clinical settings & indicate that ADSCs- enriched fat transfer could render lipofilling a reliable alternative to major tissue augmentation, such as breast surgery as augmentation, reconstruction specially in radiated breasts or secondary breast surgeries to correct complication from previous breast surgeries.

However, this protocol is not a one-step procedure but 2-steps, the first one is taking small amount of your own body fat under local anesthesia & send it to a licensed biological lab, with full accreditation  to culture & bank the mesenchymal stem cell that has prepared from your adipose tissue. The cultured ADSCs will mixed with aspirated fat, day of surgery before re-inject the enriched fat again in the recipient area.

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