Short Notes in Plastic Surgery

October 15, 2019

Chapter 73: Fat Grafts

Filed under: Chapter 73 — ravinthatte @ 6:43 am

Chapter 73: Fat Grafts

Introduction

The ability to successfully transfer fat as a free graft had eluded plastic surgeons or for that matter surgeons for nearly a century or more, though it was obvious that the tissue would be an excellent material as a filler. This blog was begun in 2011, at which time the transfer of fat was more a possibility than a reality – certainly in India – and the subject was therefore not included in its contents. Since then, in a matter of eight years, the procedure of transferring free fat has become almost routine and several papers have appeared on the subject, showing satisfactory results – not yet in the long term but certainly in the intermediate term. This fact was confirmed when a meeting in Thane city, near Mumbai, in September2018 in India was devoted to free grafting of fat and was interestingly titled as “FAT IS THE FUTURE”. The compiler of this blog inaugurated that meeting organised by Dr. Medha Bhave, a plastic surgeon from the same city, and she was therefore invited to contribute to this blog on this subject.

Because the subject of free transfer of fat is comparatively new, several terms used in this chapter may be unfamiliar to the readers of this chapter. In fact, these terms may not yet be a part of standard textbooks of plastic surgery and therefore are included here at the beginning of this chapter in order that reading this chapter becomes easier and these terms can be referred to when they appear in the text  

Glossary

  1. ADSC—Adipose derived stem cell. Stem cells are the unspecialised cells which can differentiate into specialised cells to take up a required function. In simple words it is a back-up provided by nature for future internal repairs. The stem cell may remain a stem cell after division or can differentiate into specialised cells like adipocytes, chondrocytes, osteoblasts, and myocytes. Adipose derived stem cells are adult or somatic stem cells unlike foetal embryonic cells which have ability to develop into a variegated array of cells.
  2. Aliquot—A representative sample of any “larger whole” used for chemical analysis or treatment is called an ‘aliquot’.
  3. BRAVA—Device made of two suction cups for each breast with a sealing silicone gel ring around it. These cups are connected to a non-collapsible tubing which can be connected to a suction machine. The Brava device creates negative pressure around the breasts externally, loosening the skin envelope as well as the glandular tissue in the breast to create more space for accommodating the transplanted fat. It also improves angiogenesis.
  4. CAL—Cell assisted lipo-transfer.
  5. Engraftment—Take or survival of the fat graft.
  6. EPC—Endothelial progenitor cell
  7. Fat parcel—Very small quantities or tiny droplets of fat deposited in the recipient area is called a fat parcel. Fat parcels do not pre-exist in donor tissue. They are created during processing and then injected as a fat graft. The size of the parcel is an important determinant for a successful engraftment.
  8. MSC—Mesenchymal stem cells
  9. PLA—Processed lipoaspirate
  10. SVF—Stromal vascular fraction. An intact piece of fat with fibrous septae is subjected to collagenase enzyme digestion. The enzyme lyses the septae. The resultant mixture is subjected to centrifugation and filtration. The ‘cell pellet’ obtained is called SVF. This process of preparation can be fully automated.

The cells that are found in SVF are adipose-derived stem cells (ADSCs), preadipocytes, mesenchymal and endothelial progenitor cells, fibroblasts, monocytes, pericytes, and vascular smooth muscle cells. 

SVF can be mixed with “fat graft” for a better engraftment. This is called cell assisted lipo-transfer (CAL). SVF is classified as drug/biological product and requires a regulatory approval by   the FDA in USA.

The specialised infrastructure that is necessary for harvesting and preparation of SVF includes a “clean room” facility, specialised equipment, special reagents and technical capability. This limits the widespread use of SVF in clinical practice in India not to mention legal and regulatory issues.

History

  1. Early attempts

The idea of using a “fat graft” as a filler originated and developed in Germany. The concept has evolved over more than a hundred years, beginning as an en bloc transfer and evolving to current, more refined techniques.  First documented fat transfer was carried out by Gustav Neuber. He used small pieces of fat from the medial side of the arm to correct an orbital defect in 1893. Vincenz Czerny, a Bohemian German, innovatively transferred a lipoma to prevent asymmetry after removal of a breast tumour in 1895.

Syringes were recorded to be in use since the 9th century, but it was in the year 1899 that L.M. Geer of New York patented the first screw piston syringe. The first hollow needle was constructed in 1844 by Francis Rynd, an Irish physician. The hypodermic needle was developed by Alexander Wood. It was improved to ‘lock onto the syringe’ – to avoid ejection during injection – for clinical use by Charles Hunter, a British physician who propagated injectable analgesia.

It is easy to understand that in the times when both communication and commuting were in primitive stages, the first fat transfers were pieces of fat and not injections. New innovations would take a long time to be known across continents. It was during World War I (1914-1918) that fat was used for wound healing as well as for treatment of scars caused by gunshot wounds. German maxillofacial surgeon Erich Lexer and Sir Harold Gillies of England, one of the fathers of modern plastic surgery, published their experiences in “fat grafting” in monograms titled ‘Die Frien Transplantationen’ and ‘Plastic surgery of the face’ respectively based on their work during the first war.

The first fat injection is credited to Eugene Hollander (1867-1932); another German surgeon. He used the earliest, crude syringes to transfer autologous human fat-heated with ram’s fat (to impart stability to the graft). The injection of this heated liquid concoction invariably led to a painful rash for three days, which subsided uneventfully with good end results.  His work was published in 1910-12 for indications like facial atrophy, post-mastectomy defects etc.

Charles D Miller of Chicago showed some favourable results with “cannula implants” in 1926.  He injected pieces of abdominal fat with the help of a screw piston syringe to correct crow’s feet around the eyes and other facial irregularities.  He termed these procedures as “cannula implants”.  They never became popular, due to complete resorption. He used other materials such as gutta percha, paraffin, rubber as well. His book — “Review of implantation techniques in aesthetic surgery” was criticised and derided as a “wild rambling” and without any scientific value. He was dubbed as the father of modern cosmetic surgery by some while others dismissed him as an unabashed quack.

2. Dark era

Despite depiction of many early good results in these books, fat as the ‘golden’ material started losing its sheen by 1930s as the long-term results turned out to be far from satisfactory. Surgeons soon realised that the transplanted fat shrunk, became hard and inelastic and did not survive over a long period. The purpose of achieving a ‘soft fill’ appeared to have been defeated certainly in the long term.

Lyndon Peer from Newark studied the “Loss of volume and weight in human fat grafts” in his own patients, when he evaluated their one-year outcomes. His article in the March 1950 issue of PRS indicated that fat grafts lost nearly 50% weight and volume after 1 year.  The commonest problems were formation of oil cysts and fibrosis. He also established that adipocytes survive by formation of vascular anastomosis from the recipient area which occurs around the fourth day.

Simultaneous developments were taking place in the old crude device — “syringe”. The Glass syringe was patented in 1946 by Chance brothers from England. Later, disposable plastic syringes invented in 1956 by Colin Murdoch from New Zealand became commercially available.  Despite these improvisations, near complete resorption of the grafted fat within a matter of weeks was reported.

3. Rekindling of interest

After a lull lasting for a few decades, interest in fat grafts was rekindled when Giorgio and Arpad Fischer of Italy introduced liposuction in 1977.  The technique of lipo-aspiration with safer blunt cannulas was popularised by Y. G. Illouz and Pierre Fournier both from Paris, France. Over enthusiastic suction could result in undesirable contour irregularities. These could be corrected in the same sitting with readily available, freshly sucked out liquid fat. The first ray of hope dawned when Abel Chajchir of Argentina reported first successful series of fat grafting in 1980. He advocated careful handling of the fragile graft cells to prevent cell rupture, washing the aspirate with saline to eliminate debris and establishing close contact of the fat graft with the vascular bed. The principles of the operation of “free fat grafting” laid down by him still hold true even today.

4. Understanding failures

American plastic surgeon Sydney Coleman systematised the fat grafting procedures and extensively propagated them among colleagues. His recommendations were as follows:

He advocated use of 3 mm blunt cannula and a 10-cc syringe at low negative pressure to harvest the fat with minimum trauma to adipocytes. The aspirate should then be centrifuged at a low speed of 3000 RPM to separate the oily, aqueous and cellular debris. The clean adipocyte component should then be placed in multiple tunnels made in a well vascularised recipient bed using 18 g cannula to establish a close contact with the tissues. The emphasis on the use of gentle techniques of harvesting, processing and injecting to reduce resorption significantly improved the predictability of the results.  He combined liposuction and fat grafting — a procedure he called “Liposculpture”— to refine results of body contouring. His seminal contribution and later his participation in teaching activities helped popularise “fat grafting” across the globe.

5. The dawn of the modern era

Dardick et al identified preadipocytes capable of differentiating into multiple cell types in 1976.  A team of plastic surgeons and researchers in Pittsburgh, led by J. William (Bill) Futrell, discovered that adipose tissue is a store house of mesenchymal stem cells with regenerative capabilities. They described stromal vascular fraction or SVF which is rich in adipose tissue derived stem cells (ADSCs), macrophages, fibroblasts, pericytes and the vascular endothelial progenitor cells with potential to replace the damaged adipocytes. Stromal vascular fraction is prepared by subjecting adipose tissue to enzymatic digestion to isolate the stem cells that are capable of regeneration and repair of tissues. This advance at the beginning of new millennium opened doors to new concepts in regenerative and reparative applications of the adipose tissue.  In 2004 Zuk et al showed presence of a fibroblast cell line and mesenchymal stem cells (MSC) with regenerative potential in processed lipoaspirate (PLA). These MSCs are located in perivascular space.

The regenerative potential of adipocytes was clearly demonstrated by Gino Rigotti of Italy; in 2007 for the first time, in a series of patients with radiation damage to the skin. Since then fat grafting has been widely used in post burns contractures for scarred and hyperpigmented areas and non-healing wounds to improve the quality of skin and promote healing. The interest in regenerative aspect of fat has been widening since then.

Thus, new vistas opened in the field of fat grafting to lead to the era of modern fat grafting with numerous possibilities of treating variety of clinical conditions which were difficult to treat at the time

Another milestone was added by Kotaro Yoshimura of Japan who demonstrated the mechanism of survival of fat graft with elegantly conducted animal experiments.

Current concepts in fat graft survival

Two theories have been proposed to explain the “take” of a ‘fat graft’ or Engraftment.

  1. Cell survival theory: Formation of new vascular connections from the recipient or host bed with the ‘fat graft’ is a proven mechanism (involved) in the survival of the “fat graft”. The initial phase after fat grafting involves survival by way of plasmatic imbibition up to 2 mm and then penetration of capillaries into the grafted parcel (establishment of microcirculation.) This is no different from what happens to a skin graft—as remarked by Lyndon Peer in his paper—“The neglected fat graft”, published in 1950. This paper explains why multiple tunnels, multiple planes and the tiny parcel size of the grafted fat are associated with better retention of volume (of the “fat graft”.) Immediately following the procedure of grafting; the host histiocytes go to work as scavengers to clear away damaged cells and this reflects as loss of volume and weight in the grafted fat. This is more pronounced in a traumatised graft a subject covered in this section later in the chapter while dealing with the technique of harvesting of fat for grafting. The grafted fat goes through four stages over a period of almost one year to attain a final stable result. These stages are inflammation, neovascularisation, remodelling and maturity.
    However, this theory cannot explain the other clinical effects of ‘fat grafting’ which include softening of scars, reduction in hyperpigmentation and improvement in the quality and thickness of the skin under which fat is grafted.
  2. Cell replacement theory: It was shown by Kotaro Yoshimura of Tokyo, Japan that most of the adipocytes die when exposed to ischaemic conditions after “fat grafting”. Nevertheless, ADSCs can survive and then replace the dead cells. Only the adipocytes located within 300µm get vascularised by establishment of vascular connection from the host bed. The repair and regeneration of the dead adipocytes with ADSCs occurs from day 3 to day 7 after the (fat) grafting procedure.
  3. Yoshimura and colleagues demonstrated in further studies that combination of both the mechanisms is responsible for survival of “fat grafts” under these circumstances

The UNIFIED THEORY provides the most plausible explanation of the sequence of events and outcomes in a ‘fat graft’ procedure. The conceptual possibilities are given below. 

Three zones can be identified in a fat graft parcel as shown in the diagram.

The cells in the central necrotic zone die and get eliminated within first three months. The products are cleared by host site histiocytes. Excess products would result in formation of fibrotic tissue or oil cysts by the end of three months. Three to nine months is the period of stabilization of fat graft, when the cysts wall would be consolidated or calcified. The peripheral zone survives. Enhancing neovascularisation using various methods can help better survival of this zone. We will see the details later.

The dying fat cells in the middle zone are replaced by ADSCs that differentiate into adipocytes. This is aided by the angiogenic factors released by ADSCs. One can clearly see why cell assisted transfers, that add more ADSCs by way of SVF result in better retention of the graft.  The SVF provides good number of ADSCs to replace the dead adipocytes in the fat graft.

Summary of events in a fat graft can be represented as follows:

Thus, a period of 9 months to one year is necessary to evaluate the final outcomes of a “fat grafting” procedures.

In summary and based on empirical evidence the factors that result in better take of the grafted fat and ensure better long-term survival are as follows

  1. The harvest should be as less traumatic as possible preferably with a large cannula and is done following infiltration of saline with or without appropriate amount of adrenaline. This is called the wet technique
  2. The harvest is best performed using low pressure to avoid trauma
  3. From the time that the fat is harvested little time should be wasted before it is injected
  4. The bed into which fat is injected should be vascular
  5. The parcel size of the graft should be small
  6. The parcels should be deposited in multiple tunnels in different planes
  7. The injected material should include copious amounts of SVF
  8. The capacity of the area into which the fat is introduced must be gauged and excessive injection is avoided because any pressure on the graft within the tissue planes leads to local ischemia

Indications

The improvements in results of “fat grafting” due to a better understanding of the mechanisms involved in the take of grafts as well as refinements in the techniques have resulted in a paradigm shift in the treatment of some defects routinely seen by plastic surgeons

 Fat can be used in two ways. It can be used as a filler to camouflage or fill a defect for cosmetic or reconstructive purpose. Alternatively, the paracrine effect of fat can be used for regenerative purposes. This effect consists of both angiogenic as well as a neurogenic element in addition to the stem cells that derive from the adipocytes and which can differentiate into various types of cells.  The two basic indications for use of free fat as a graft are as follows

  1. Structural fat grafting: Fat graft is used as a filler. Fat is an ideal filler because it is autologous, biocompatible and integrates naturally into the local tissue. The material is abundant and therefore available for repeated use. Fat has been used successfully as a filler in the face, breasts, buttocks, and various types of depressed and contracted scars in various parts of the body 
  2. Fat graft as regenerative material: Fat is an active and dynamic tissue as opposed to what has been understood in the past containing many “cell types” with different functions in addition to those of the stem cells. Fat is used for its angiogenic potential as well as stem cell content which bring about tissue regeneration and helps accelerate wound healing. More importantly, the ADSCs in this procedure are easy to harvest in larger numbers as compared to ‘bone marrow stem cells.’’ A list of indications given here is not exhaustive in detail but merely represents those conditions in which clinical success of fat grafting has been successfully demonstrated. The potential of fat grafting procedure appears to be unlimited and not yet fully explored. Objective assessment of the results is difficult to carry out especially in the procedures done with a regenerative potential as the goal.  Histopathological evidence of changes is a reliable tool to prove the success of the procedure but is difficult to use in clinical practice, while animal experimental facilities are not easily available to clinicians and are also difficult to conduct for conditions that are specific to human skin—for example—hyperpigmentation.

FACE—In cases of congenital defects like hemifacial atrophy fat may in due course of time replace muscle transfers and dermal grafts. A fat graft creates softer contours and far better symmetry than the former when successful. Management of age-related facial changes have been refined with the addition of fat grafting to restore the volume that is lost in the process of ageing. Fat atrophy also significantly adds to sagging. Therefore, instead of just removing and balancing the lax skin, the procedure of filling up the area with “fat grafts” restores more natural contours, avoids the stretched look that resulted following the older techniques. Additionally, stem cells and the paracrine effect of the adipocytes brings about neo-angiogenesis and leads to improvement in dermal thickness and improves actinic damage and pigmentation. Fat is also used as a regenerative material to improve a wide range of aesthetic problems like dull, aged, thinned, sun-damaged skin, hyperpigmentation of face and hands. Used in this manner, the dermal thickness and colour improve. Among non-aesthetic problems, morphea and scleroderma are some of the conditions that have been shown to improve on similar lines with “fat grafts”

BREAST—Fat transfer to the breast in congenital asymmetry, small breasts, and correction of secondary asymmetries after implants has met with variable success. Dr. Roger Khouri of Miami described a technique for natural augmentation of breasts with fat grafting preceded by application of a vacuum Bra to the breasts (BRAVA) for a period of about 3 months in order to improve vascularity and loosen up tissues for a large volume transfer in one session. Without this preoperative expansion a transfer of more than 150 ccs in one sitting is not feasible. Without BRAVA, the patient requires 2-3 sessions of 150-200 cc each. The downside of using fat graft for breast augmentation as a substitute for implant is that the forward projecting, turgid youthful breast cannot be recreated as is possible with implants. Usually a “fat grafted” breast appears snoopy and ptotic. The other concern is that of micro calcification of necrotic fat which may interfere with a radiological diagnosis of malignancy. In the recent past breasts reconstructed with expanders have been treated with fat grafts in stages in which fat is introduced by gradually reducing the volume of the expander the volume of fat being equal to the reduction of the volume of the expander.

Excellent results have been demonstrated by Dr. Gino Rigotti in patients who require reconstruction after radical mastectomy for cancer of the breast but the results of fat transfer to breast are elusive and consistent results have been demonstrated by very few surgeons.

Fat grafting is also useful after radiation damage to the breast.

BUTTOCKS—Another use of fat that has gained popularity is in augmentation of buttocks by fat transfer. Fat embolism is not uncommon after this surgery. Avoiding injection into gluteal muscles, limiting the fat deposition to subcutaneous planes, avoiding puncturing deep veins under the muscle and avoiding over-injection to prevent entry of fat into thin walled sub gluteal plexus are the safety measures that have been suggested to prevent complications.

SCARS—Acne scars and post traumatic depressed scars of the face, nose and other areas can be improved with subscision with a needle to release the scar sub-dermally and filling with fat. Controlled trials are needed in order to assess efficacy of this process in comparison with other modalities like derma roller, fractional lasers and radiofrequency needle ablation. Painful and contracted scars improve with fat grafting due to neo-angiogenesis and neo-collagenisation brought about by the “fat graft”. Pain relief can be attributed to release of nerve entrapment. Demonstrable increase in scar elasticity and suppleness, improvement in colour and volume deficit and improved mobility of body parts are the advantages of fat grafting under a painful scar. Post-burns scars can benefit with multiple sessions of “fat grafting” under them.

Chronic wounds in diabetes or varicose veins, pressure sores, radiodermatitis benefit from the stem cell content of the fat graft by promoting healing due to their angiogenic effect and presence of stem cells.

Lack of better levels of evidence is a major obstacle in use of fat as regenerative material in routine clinical practice. While exploring more uses of fat grafts is warranted, better ways of demonstrating clinical improvement and graft take need to be devised.  This is because the effects like improved dermal thickness, elasticity, softness, and reducing pigmentation are not easy to measure objectively with the help of simple measuring techniques and photography.

Anaesthesia

The anaesthesia for cases of ‘fat grafting’ depends upon the volume of fat that needs to be transferred and the status of the recipient area. If the amount to be transferred is large; a regional or general anaesthesia in the form of either total intravenous anaesthesia (TIVA) or a complete GA with either intubation or a laryngeal mask is used. It should be remembered that infiltration of the donor area with fluid together with a local anaesthetic agent for harvesting the graft itself can be painful due to stretching of tissues. Therefore anxious patients with a low pain threshold may need sedation. Rigottomies or subscision in the recipient areas as well as and injections can also cause pain. Infiltration of recipient area with a local anaesthetic containing lignocaine can be detrimental to survival of the graft as will be described in the following section where a method to reduce this effect by a wash will be described

Techniques of fat grafting

The key to successful ‘fat grafting’ lies in the methods used both in its harvest and its implantation. A good technique ensures that the harvest and injection of fat do not cause any damage to the fat cells, the impurities in the form of RBCs and other cells and the debris as well as the floating supernatant oil that can cause inflammation are removed.  This is called ‘processing’.  The ‘bench time’, that is the interval between harvesting and injection which is utilised for processing must be minimised. In addition, the recipient bed must provide adequate or good vascular support to the grafted fat for it to ‘take’ (engraftment) as well as for the replacement of dead cells by pre-adipocytes and other stem cells. With these basic principles in mind, innumerable variations have been described in the techniques used by various surgeons for this procedure.  It must be remembered that the final outcome is the net result of graft injury and vascularity available at the recipient bed irrespective of the permutations and combinations of the various methods used. For these reasons, there is no single best technique among the many described that ensures good uptake of the graft every time and it is the interplay of various steps in the hands of a particular surgeon that determines the final result.

Basic steps—We can consider a fat grafting procedure as a series of steps which are— harvesting, processing, the preparation of the recipient area, technique of injection and last but not the least ‘cell assisted enhancements for a better take of the ‘fat graft’.

A-1—Harvesting the fat—The degree of trauma to the fat graft during harvesting is the most important factor that influences the take of the graft. The lesser the trauma the better is the ‘take’.

I – donor area — The area that can be suctioned without leaving behind a visible deformity is selected. An aesthetically pleasing contour of the donor area should be achieved post-operatively. It should preferably be located in the same operative field as the recipient area for convenience.  Common areas chosen as donor areas are the abdomen and the thighs. There is no difference in the quality of graft harvested from different areas but empirically fat from the thigh is used when structural stability of the graft is desirable i. e the graft is used as a filler. Also, empirically, the abdominal fat is believed to produce a better angiogenic effect.

II Canula — Cannulae are used for harvesting the fat as well as injecting the fat. Blunt cannulas of diameter between 3 to 6 mm are commonly used to harvest the graft. Best graft viability is documented with 6 mm cannula.

Fat harvested with bigger cannula and lower suction pressure can be subjected to higher centrifugation speed to separate blood and debris.

Blunt canulae are used for injecting the fat. For structural fat grafts at a deeper level and larger amounts of injection, 3 mm cannulae are used. This is required for fat grafting of breasts and buttocks. For structural grafting at a more superficial level and smaller quantities at a deep level as in the face, a one mm cannula is used. A 0.7 mm cannula is used for ‘nano’ fat grafting where surviving adipocytes are practically absent, and the goal is to inject stem cells and preadipocytes for rejuvenation. The number, type and distribution of holes in the cannula that suck the fat is variable. Usually 1 to 3 blunt holes distributed evenly around the perimeter of the cannula near the tip is the preferred option. Some surgeons use a nine-hole cannula to prevent injury to the fat during its passage through the cannula.

III Aspiration devices — Many suction devices based on the principles of suction can be used for harvesting fat grafts. These include a simple syringe, as well as power / water jet / ultrasound assisted liposuction devices. Specially designed devices with cell concentrating mechanisms can also be used for aspiration. Though these are expensive, they have not shown great improvements in cell counts. Whichever device is used, low negative pressure during aspiration saves adipocytes from injury due to implosion. The speed of harvesting drops when low pressure is used. This is inconvenient when large amount of fat is required as in cases of enhancement of breasts and buttocks. High negative pressure used in liposuction results in disruption of 90% of the fat cells. In such a situation, moderate negative pressures combined with the wet technique, which will be described in the next para, and purification with a manual centrifuge can overcome the detrimental effect of high power suction and produce satisfactory results.

IV – Techniques of harvesting — The fat can be harvested with dry or wet techniques. In the dry technique, fat is aspirated without prior infiltration. This improves yield of stem cells due to injury to the vascular channels around which the stem cells are located. But the survival of the fat cells is less satisfactory than in the wet technique. In the wet technique, normal saline or Ringer’s lactate solution along with adrenaline as a vasoconstrictor to promote vasoconstriction and lignocaine as a local anaesthetic is infiltrated into the donor area before the suction begins. This is also called tumescent technique. Tumescent technique is the preferred technique world-wide. The dry technique is rarely used nowadays.

Another technique described by B. Guyron is core fat grafting. Tiny cylinders of fat are cored out with the help of an obliquely cut insulin syringe advanced in a rotating manner into the donor area through a 5 mm incision. The piston of the syringe is pulled only to provide space for the graft in order to enter the syringe. The graft thus obtained is transferred to the recipient area prepared by sub-cutaneous dissection through a short incision. The pushing of the piston introduces the fat core into the recipient area. This technique ensures excellent “graft take” due to preservation of the fat cells. But due to the tedious nature of the technique, it is reserved for small areas. Excellent predictable results have been demonstrated with this technique.

A-2—Processing

The harvested fat graft is processed in many ways to improve the graft take. The processing is aimed at removal of impurities. These impurities consist of RBCs, collagen fibres, lignocaine and broken and injured fat cells. Blood and debris cause inflammation and fibrosis jeopardising the cell survival. The fibrous tissue can be an obstacle while injecting the graft. This results in blockage of syringe, increased injection pressures and damage to the adipocytes. Lignocaine is usually part of the infiltrate used for harvesting the graft. It is known to hamper the viability of the adipocytes and should be rinsed away. The lipids released by the broken adipocytes can also trigger inflammation and fibrosis. These undesirable components of the graft invariably result in false estimation of volume being injected and is responsible for the delayed loss of volume of the graft. The processing also aims to improve concentration of adipocytes in the graft.

The various methods of used for processing are centrifugation that may be manual or automated, decantation or sedimentation, washing, filtration and syringing,

The harvested fat graft can be first washed to remove traces of lignocaine and blood by adding a small volume of normal or physiological saline and removing it by any of the above methods.

Centrifugation is the most widely preferred method. The most popular protocol was suggested by Coleman. He advised centrifugation at 3000 RPM for 3 minutes. The graft then separates into three layers. The topmost layer is made up of oil and broken fat. It can be either poured out or absorbed with a wick of cotton. The middle layer consists of injectable fat tissue. The lower most layer containing infiltration fluid, RBCs and debris can be ejected out from the end of the syringe (please see figures at the end of the chapter).

Centrifugation force of more than 50 g is detrimental to the survival of adipocytes. These damaged adipocytes release lipids and then undergo apoptosis after transfer. The angiogenic potential of the graft suffers; as tubule formation — which is essential for vascularisation of the graft and further nourishment for survival — is hampered. Excess speed also increases fluid separation resulting in reduction in the injectable component of the graft and increased chances of formation of oil cysts.

When large volume “fat grafting” is done, it may be collected in flexi-bags which are hung to decant. The fluid is let out and graft is collected in syringe for injection. The topmost layer of oil is discarded. The decantation has been proven to be inferior to centrifugation.

Many studies have been conducted to compare various methods. The filtration has been shown to cause more nodule formation than centrifugation.

Condensation of the graft — After carefully understanding the mechanism of graft survival, one can appreciate that after the initial phase of survival of the cells in the peripheral zone, the dying adipocytes in the regenerative zone are replaced by pre-adipocytes and stem cells in the graft. Thus, the graft should contain a fair proportion of pre adipocytes and stem cells. In order to reduce the number of mature adipocytes—to reduce competition for blood supply — and increase the relative number of stem cells—to replace the dying adipocytes, the graft is mechanically agitated. The harvested fat is passed to and fro between two syringes joined with a connector. This is called condensation. The oil formed during this process needs to be filtered. Dr. Patric Tonard is credited for this method in order to inject stem cells which are useful in rejuvenation of face. In his method, practically most of the adipocytes are broken, the oil is filtered and only stem cells that remain are injected.

A-3—Preparation of recipient site

The preliminary requirement for graft survival is a vascularised recipient bed. The pressure created by the graft due to space occupied by it can also compromise vascularity. In olden days, in order to compensate for the likely graft loss; injecting fat in excess of the estimated requirement was practised. But this concept was found to be incorrect. The increase in the tissue pressure brought about by the excess graft actually causes more graft loss due to vascular compression. Thus, increasing the number of sessions rather than over-injection of fat was found to be conducive to better graft take.

In order to improve the graft, take, in scarred beds an innovative method was developed by Dr. Gino Rigotti. It came to be called Rigottomy. He uses a thick needle to break the scar with a snapping movement of the needle to create space for the graft. This in turn reduces the tissue pressure that would otherwise build due to injection into tight space. This is very useful technique in post mastectomy breast reconstructions with fat graft, burns scars and acne.

Some innovative methods were described with use of negative pressure in cases of breast enlargement with fat.  BRAVA was a pair of silicon cups to be worn by the patient like a BRA. This is connected to a suction apparatus for certain time daily in order to loosen the breast and create space for the fat graft. The negative suction also promotes angiogenesis similar to VAC used for improving the state of contaminated and infected wounds, further improving engraftment.

A-4—Fat transferTechniques

The prepared fat needs to be transferred to the prepared recipient bed without trauma. Syringe with needles or canulae are used for smaller volumes whereas a power assisted device can be used in cases of larger volumes like breast, buttocks or large burns scars. Usually blunt canulae of size 3 mm are used for large, structural fat grafts as is required in case of breast and buttock. This is also called “macro-fat grafting”. The smaller volumes can be injected with 1 mm cannula in structural fat grafting for the face and various defects that need filling. The micro-fat grafting is an important ancillary technique in “face lifts” to address the age-related “fat atrophy”. The “nano-fat graft” in which most of the adipocytes have been destroyed and preadipocytes and stem cells are left behind is useful in facial rejuvenation, improvement in actinic or radiation damage of the skin.

“Expansion vibration lipofilling” is a technique described recently. In this technique; the reciprocating movement of a power assisted device is used to create space with a special boat shaped canula and then injecting fat to reduce the trauma to fat cells due to pressure of injection. This is particularly useful in large fat grafts.

Parcel size and its importance — The fat parcel is the tiniest drop that is being injected. It is essentially a conglomerate of adipocytes. It is important to realise that the parcels do not pre-exist in the donor area. They are created by shearing and processing at the time of harvest and injection. The bigger the parcel size the central necrotic zone will be bigger, resulting in graft loss and other complications.

B.  Cell assisted fat transferVarious substances have been added to improve cell replacement in the grafted fat. This includes platelet rich plasma (PRP) and stromal vascular fraction. The PRP contains growth factors but no conclusive evidence is available to use PRP in routine practice to improve the results. SVF as a biological product is prepared by use of enzyme collagenase to digest the fibrous tissue from an intact piece of fat tissue. The collagen is then washed away and cell pellet containing stem cells is obtained. These cells can either be cultured for injection later or mixed with fat graft and injected.

The stem cell in routine lipoaspirate are sparse as the aspiration process avoids trauma to vessels and fibrous septae that separate the fat compartments. The stem cells are located near the vessels. Hence it is necessary to take a separate piece of intact fat for isolation of SVF. SVF assisted fat transfers exhibit better volume retention, viability and vascularity of the grafts. Since the use of the enzyme collagenase is frowned upon due to possible allergic reactions in clinical practice, SVF fat transfer is restricted and limited to certain institutions only under FDA regulations. The Indian as well as international law regarding use of stem cells keeps updating from time to time and needs to be strictly complied with.

Complications

The commonest undesirable result of fat grafting would be a loss of the graft leaving behind inadequate improvement in the deformity. A period of one year is the bare minimum to evaluate the result of a ‘fat graft’.

  1. Complications in the donor area — These are bruising, oedema, haematoma, local infection, problems with wound healing leading to hypertrophy of the scar or keloid formation. Sometimes, irregularity of the contour of the donor area can result in aesthetic concerns. Rarely, a muscle or even the peritoneum may be punctured due to the harvesting canula.
  2. Complications in the recipient area — Early complications include infection, seroma, haematoma, swelling, bruising, injury to vessel or nerve during injection. The dreaded complication is fat embolism which will be discussed at length in a subsequent paragraph. Late complications include oil cyst formation, fibrosis, and irregularities in the areas where fat has been grafted. Complications and adverse results in specific parts of the body are mentioned below.

Breast Fat grafting of breast is commonly done for two indications viz. aesthetic augmentation and post-mastectomy reconstruction if cases of breast malignancy. The common complication of a fat graft is calcification of necrosed fat. It may be difficult to distinguish from a malignancy. In cases where a breast was reconstructed with “fat grafts ‘following surgery for malignancy fatty masses may appear after the grafting procedure which may need needle biopsies to rule out recurrence.

Face — A ‘fat graft’ in the “danger areas” of the face; such as the nasolabial folds and the area surrounding the tip of the nose; can lead to thrombosis of the ocular or even a cerebral artery. Excessive pressure while injecting especially on the nose or eye lids can cause migration of fat into the ophthalmic or even the internal carotid artery. It is imperative to check for the absence of a back flow of blood while injecting and use blunt tipped cannulas instead of needles to avoid such dangerous complications. Blindness has been reported after fat grafting in the face.

Buttocks — the Brazilian ‘butt lift’ includes injection of very large volumes of fat into the buttocks as an ancillary. The recent ISAPS (International Society of Aesthetic Plastic Surgeons) guidelines clearly warn against injection into the gluteal muscles. Such injection results in building of tissue pressure and migration of fat in the thin walled sub-gluteal plexus of vessels leading to fat embolism. The injection into the buttock must be restricted to subcutaneous tissues. Fatalities have been reported on account of ‘fat embolism from this area following faulty techniques. 

Controversies in ‘fat grafting’

The results of fat grafting’ are linked to an interplay of various steps. The exact method that guarantees a reasonably predictable ‘graft take’ has eluded the plastic surgeons. There are numerous studies with conclusions which contradict each other. For example, some studies say that there is no difference in results irrespective of whether the graft is subjected to centrifugation, sedimentation or filtration; while some studies conclude that the centrifugation is the best method for processing the graft.

 The cell assisted fat transfer has both proponents as well as opponents. Lack of good laboratory facilities limits the use of this method in most clinical centres.

Innumerable substances have been added to help graft take but there is no conclusive evidence to support any particular one substance.

The exact amount of fat that needs to be transferred in view of the possible loss of the grafted material is difficult to predict objectively.

As this is a comparatively recent and also a rediscovered technique with some promising early results, the long-term results, especially complications are yet to be fully assessed. An occasional case of hypertrophy of the grafted fat that needed excision has been reported.

The methods to assess results are either physical methods or by way of imaging techniques or a histopathological study of the grafted material. None of these have been used objectively in humans in a large enough study to draw any definite conclusions. Available studies are either animal experiments or consist of a limited human series.

There are legal restrictions on the use of stem cells and the changing laws need to be followed to avoid legal issues.

The future — The technique of ‘fat grafting’ is slowly unfolding before us with efforts of researchers and plastic surgeons, throwing newer light both on the technical as well as the physiological aspects of this procedure. Fat is surely a good source of multi-potent cells that can be used to treat various deformities in different parts of the body and its main attraction is its simplicity; yet its effectiveness is not yet proved beyond doubt.  A few conditions like tough, hyperpigmented, scarred, damaged skin can be treated successfully unlike in the olden days when the conditions did not have any remedy.

Lab grown fat can be a source of unlimited multipotent cells which may find various uses in future. Given the right environment, these cells may be used to create parts like cartilage framework for an ear and can also be used as a dorsal strut for the deformity of a saddle nose.  It has already shown great promise in breast reconstruction. Widespread use and critical analysis of factors involved in successful fat grafting as well as the long-term results will decide the future application of this intriguing as well as promising technique.

Pictures contributed Dr. Medha Bhave, Mumbai

Pictures contributed by Dr. Nitin Mokal, Mumbai

Pictures contributed by Parag Sahasrabuddhe, Pune

Several weeks after this chapter (contributed by Dr. Medha Bhave) was uploaded, Dr. Durga Karki, Professor, Dept. of Burns & Plastic Surgery, Delhi, presented a paper on the subject of “Lipofilling – A Regenerative Alternate for Remodeling Burn Scars: A Clinico-immunohistopathological Study” which was the subject of an earlier paper for which she won the Kilner Essay award of the Association of Plastic Surgeons of India. This paper was presented as a part of the annual conference of the association in the plenary session by invitation. While the literature is replete with effects of stem cells on tissue remodeling, Dr. Durga’s paper perhaps for the first time in India presented actual histological evidence of the remodeling of the scarred skin in cases of burns following injection of fat which presumably included stem cells though the presenter did not lay claim that she had transferred stem cells specifically. The photographs of the histology slides are reproduced below with their legends with thanks to Dr. Durga Karki.

HEMATOXYLIN & EOSIN STAIN (100x)
A: Pre treatment, B: post treatment
Collagen fibers became more organized and thin compared to pre treatment level (large arrow).
Focal reduction in melanocytes in post treatment stain (small arrows).
MASSON TRI-CHROME (200x)
A: Pre Operative B: Post Treatment
Significant deposition of fine collagen fibers at reticular dermis (arrows) seen in post treatment stain compared to pre treatment stain
VERHOEFF ELASTIN STAIN (200x)
A: pre treatment, B: Post Treatment
Amount of elastin fibers increase significantly and organization of fibers improves (arrow) seen in post treatment stain compared to pre treatment stain
p-53 (400x)
A: Pre treatment, B: Post Treatment
Increase expression of p-53 in basal layer of epidermis in post treatment stain compared to pre treatment stain
S-100 (400x)
A: Pre treatment, B: Post Treatment
Reduction in expression of S-100 in basal layer of epidermis in post treatment stain compared to pre treatment stain

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