Histopathologic eva Iuation of adipose autografts in a rabbit ear model JEFFREY BARTYNSKI, MD, MITCHELL S. MARION, MD, and TOM D. WANG, MD, Rochester, Minnesota
Injectionof autologous adipose tissue removed via liposuctionhas been used clinically for facial contouring, the aging face, furrows, facial atrophy, acne scars, nasolabial folds, chin, and various other surgical defects. Survival rates for autografts of fat have been quoted anywhere from 30% to 80%. Our study uses a reproducible rabbit animal model for autotransplantation of adipose tissue and examines the histopathologic changes that occur to the graft over time. Autogenous subcutaneous fat was removed from a dorsal scapular donor site, treated to stimulate cannula damage as in liposuction, then relnjected at the base of the ear. Histologic examination of the grafts were made at 5,10,15,20,40, and 100 days after transplantation. Hematoxylin-eosinsections were graded on degree of fibrosis present (0 to 4+), viable fat (1 to lo), degree of inflammation (0 to 4+), and neovascularization ( + or -1. Viability of fat decreased from 8.5 to 10 at 5 days to 2 viabillty at 40 days. Acute inflammation peaked at 10 days, followed by the chronic Inflammatory response with macrophages and multinucleated giant cells scavenging the dying fat graft. Neovascularization began at 5 days, peaked at 10 days, and remained constant thereafter only at the edge of the graft. Microcysts appeared at 15 days and Increased in number in proportion to the decrease in viable fat. In summary, the temporal histologic events are progressive fibrosis; decreased amount of viable fat; inflammation beginning with a neutrophillc response, later a macrophage and giant cell response; and neovascularization at the periphery of the grafl insufficient to maintain graft viability. In our animal model, autografts of fat appear to have limited long-term viability and are replaced by fibrous tissue. This may have clinical implications in autografting of fat in facial plastic and reconstructive Surgery. (OTOIARYNGOL HEAD NECK SURG 1990;102:314.)
I n the practice of facial plastic and reconstructive surgery, correction of soft tissue defects is a common and often difficult procedure. The perfect soft tissue substitute is yet to be discovered. Various synthetics, including paraffin, liquid silicone, encapsulated silicone, silicone rubber, plastic, and various collagen extracts have been tried. Recently autogenous transplantation of fat obtained via liposuction has gained favor among facial plastic surgeons. 2-6 Historically, the first use of free autogenous fat transplants was described by Neuber in 1893.7 He transplanted small pieces of upper arm fat to correct a depressed facial scar. In 1895, Czerny' used a large lipoma to fill a breast defect.8 Later, in 1911, Tuffier' described
From the Department of Otolaryngology, Mayo Clinic. Presented at the Annual Meeting of the American Academy of Otolaryngology-Head and Neck Surgery, New Orleans, La., Sept. 24-28, 1989. Submitted for publication Sept. 26, 1989; accepted Oct. 30, 1989. Reprint requests: Jeffrey Bartynski, MD, Department of Otolaryngology, Mayo Clinic, 200 1st St., NW, Rochester, MN 55905. 2311118434
pathologically the outcome of transplanted fat in the extrapleural space. The material was examined 4 months after transplant and found to be absorbed and replaced by fibrous tissue. In 1912, Zipper" examined microscopically free fat transplants after two mammoplasties and found encapsulated fat, as well as normal fat. Peer" performed a classic series of experiments in human beings in 1950. Thirteen cases of transplanted fat were moved in large blocks. Another thirteen subjects received the same weight of graft, but in smaller pieces. All fat was transplanted into the rectus abdominus sheath. The fat was later removed and examined histopathologically. The results revealed that neovascularization begins by the fourth day after transplantation and that there is some graft liquification. However, only about 45% of the original volume was lost in I year. He concluded that autotransplanted fat does indeed survive, refuting theories that histiocytes would absorb the fat and replace the graft. The next several decades saw the advent of synthetic implantable material gain widespread favor for implantation with its easy availability and lack of secondary donor-site problems. Then, in 1977, Illouz12introduced
314
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Histopathologic evaluation of adipose autografts 315
the technique known as liposuction. Initially used as a procedure to rid the patient of aesthetically unpleasing fatty deposits, it also provided the facial plastic and reconstructive surgeon with an abundant supply of potential graft material. In 1984, Bircoll13 first presented his technique of injecting autologous adipose tissue removed by liposuction. He described fat injection in facial contouring and for treatment of the aging face, facial furrows, midfacial atrophy, breast reconstruction, and cosmetic chest augmentation. Transplanted fat pearl autografts were resurrected by Ellenbogen* when he reported his experience with acne scars, nasolabial folds, chin, and eyelid depressions. In 1987, Newman and Levin3 summarized their experience with facial lipotransplant surgery, injecting such sites as melolabial folds, malar region and inframalar groove, forehead and glabella, and the mentum. The widespread clinical use of autologous fat obtained by liposuction for transplantation has progressed at a much faster pace than basic research. Actually, little basic science work has been done specifically looking at autologous fat transplantation. Survival rate for transplanted fat has been reported anywhere from 30%14 to as much as 80%.15 Methods of assessing adipose tissue viability are mostly performed on either a volumetric basis or from histologic study. ' 1 . 1 4 . 1 b . 1 7 Animal models have been used for various autografts and allografts. An excellent model to study implantable materials is the rabbit ear model of Breadon, Kern, and Neel. From their papers, histologic cross sections of the normal rabbit ear demonstrate no endogenous fat. Thus it would seem that the rabbit ear would be an ideal location to test autografts of fat. METHODS
A modification of the rabbit ear model used by Breadon, Kern, and Neel was used for this study." New Zealand white rabbits were each injected with autografts of fat at the base of each ear. Each rabbit was followed for a distinct predetermined period of time and cared for in accordance to the guidelines set forth by the institutional animal care committee. Anesthesia was induced with 35 mglkg of ketamine, mixed on a 9: 1 ratio with xylazine, then injected intramuscularly into the lateral thigh region. Each rabbit was given 600,000 units benzathine penicillin G intramuscularly in the hind leg before the procedure. A 5 x 5 cm paraspinal (interscapular) donor site was shaved, cleaned with thimerosal and 85% ethyl alcohol, and draped in a sterile manner. The area was infiltrated with 5 cc of I % lidocaine 1 : 100,000 with epinephrine (wet technique). The fat was harvested as follows: under sterile conditions a 1.5 cm skin incision overlying the fat pad was made with a #15 blade. The fat pad
was then sharply dissected with scissors and harvested. This fat packet was minced with scissors into a petri dish containing lactated Ringer's solution and cut into approximately 1-mm pieces. These pieces of fat were transferred into a 10-cc h e r lock syringe with control rings and passed through a 3.3-mm accelerator aspiration needle and cannula (Byron Medical, Tucson, Ariz.) to simulate cannula damage in liposuction. Fat to be injected was placed into a microinjection gun (THEGUN, Byron Medical, Tucson, Ariz.), which injects 0.1 cc with each click. An 18-gauge blunt-tipped needle with introducible trocar is attached to the tip. The rabbit ear recipient site on the dorsal (convex) surface was shaved, cleaned with thimerosal and 85% ethyl alcohol, and draped in a sterile fashion. The GUN, with 18-gauge blunt-tipped needle and trocar attached, was tunneled subcutaneously until the tip of the cannula rested 1 cm from the base of the ear. Fat obtained by the above method was split into equal sample volumes and injected into each ear, raising a bleb. Volume necessary to raise the bleb was recorded (usually 4 cc). The graft was observed for any signs of rejection (i.e., erythema, induration, and/or skin slough). The rabbits were killed by a lethal injection of pentobarbital at 5 days, 10 days, 15 days, 20 days, 40 days, and 100 days after transplant. The ears were removed and preserved in neutral buffered formalin. After the grafts were carefully dissected from their bed, serial sections were cut, imbedded in paraffin, and hematoxylin-eosin stains made. Representative sections were examined histologically under 20 x magnification. Ten random microscopic fields were examined and graded on degree of fibrosis (0 to 4 + ); viable fat (0 to 10); inflammation (0 to 4 + ); and neovascularization ( + or -). Predominant inflammatory cell type, multinucleated giant cell response, and microcysts were observed and their temporal relationship noted. RESULTS
Distinct histopathologic events occurred over time in the rabbit fat autografts. In the grafts examined at 5 days, the degree of fibrosis was very minimal and rated 1 . A very thin capsule was noted surrounding the graft and adherent skeletal muscle was noted. Viability of the fat appeared histologically to be very good, with normal fat architecture and normal adipocytes throughout the graft. Using our rating system, the viability at day 5 was rated anywhere between 8.5 and 10. Examination of the inflammatory response revealed a 1 + to 2 + response. The predominant inflammatory cell type seen was neutrophilic; a very limited number of macrophages were encountered. Most of this inflammatory response occurred at the periphery of the graft. Neovascularization was noted to occur in fibrous septa
+
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316 EvWTYNSKI et 01.
P
.
*
s,
d
L
i.
Fig. I . Day 5.Skeletal muscle bed with thin capsule surrounding normal adipose tissue [autograft).
Fig. 2. Day 5.Normal fat with neovascularization noted along graft edge.
near the periphery only, with the center of the graft remaining avascular (Figs. 1, 2, and 3). Ten days after transplant, the fibroblastic response was rated 2 + , with thickening of the surrounding capsule and increased fibrous septa. Histologic viable fat decreased to a 7 rating, with fat necrosis and collapse of the adipocyte walls encountered in the center of the graft. Acute inflammation peaks at 10 days at 3 + , with numerous neutrophils seen engulfing the adipocytes.
The beginning of the chronic inflammatory response is noted with macrophages accumulating, lipid near the graft edges. Few multinucleated giant cells are also first encountered at this time. Neovascularization peaks, but only at graft edges (Figs. 4, 5, and 6). The graft at 15 days shows little change from 5 days earlier, with a similar fibroblastic response and a viable fat rating of 6. An inflammation of 3 + is seen with an equal number of neutrophils and macrophages. The first
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Histopathologic evaluation of adipose autografts 317
Fig. 3. Day 5.High-power view of neovascularizationwith neutrophils in fibrous septa.
fl-
Fig. 4. Day 10. Capillary filled with RBCs along the graft edge.
appearance of microcysts is encountered at 15 days. The importance of these cysts will be noted in later specimens. Vascularization of the graft remains constant and only at the periphery. At 20 days, the grafts demonstrate a great deal of histologic change. Fibrosis increases dramatically, with a 3 + rating and a thick capsule and islands of fibrosis throughout. A marked drop occurs in the amount of histologic viable fat, to a rating of 4. Inflammation is
now almost entirely of the chronic cellular type and 4 . Lipid-laden macrophages are seen, as well as a rise in the number of microcysts-especially where viable fat is absent. The rise in microcysts parallels the decline in viable fat. Vascularity of the graft remains isolated to the margins of the graft (Figs. 7 and 8). Forty days after transplant, fibrosis reaches a maximum rating of 4+, with fibrosis throughout the substance of the graft. Viable fat is only rated 2, with few
+
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t
Fig. 5. Day 10.Note increasing neutrophilic response on this view.
Fig. 6. Day 10.Area of exuberant neutrophilic infiltrate.
isolated “islands” of histologically viable fat. The chronic inflammatory response remains 4 + , with numerous macrophages scavenging the dying adipocytes. Also, a rise in the multinucleated giant cell (foreign body) response is noted at this time. Neovascularization remains insufficient and isolated. The graft itself is composed predominantly of microcysts and fibrous tissue when examination is performed at 40 days (Figs. 9 and 10).
Grafts examined at 100 days after transplant demonstrated little change from the histologic preparations made at 40 days. The progressive fibrosis and diminished fat viability had stabilized. A similar amount of microcysts were noted. SUMMARY The temporal histologic events occurring in autografts of fat obtained via liposuction in a rabbit model are as
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Histopathologic evaluation of adipose autografts 319
Fig. 7. Day 20. Multinucleated giant cell response noted on this view.
c
Fig. 8. Day 20. Areas of graft composed of predominantly microcysts.
follows. Graft fibrosis follows a progressive course, with an initial capsule formation followed by fibrous septationsand exuberant fibrosis of the entire graft. This fibrous reaction peaks at only 40 days after transplant and remains constant, with only slight progression noted at 100 days. Viability of the fat itself also diminishes over time. Initially on histologic evaluation, the grafts appear 80% to 100% viable. This diminishes gradually, however,
up until 15 days, and drops precipitously at 20 days to 40% viability histologically. The final viability appears to be 20% to 30%, as interpreted from our histologic preparations. Neovascularization begins along the graft periphery by 5 days. This neovascularity never proceeds past its peak at 10 days, and only occurs along the graft edges. Thus the graft never achieves an adequate blood supply at its center to maintain its long-term viability.
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Fig. 9. Day 40.Multinucieated giant cells scavenging a microcyst of fat with surrounding fibrosis.
Fig. 10. Day 40.Fibrous septa in center of the graft. Note the small vessel supplying an island of normal fat.
Inflammation was characterizedby acute and chronic phases. The acute phase, composed predominantly of neutrophilic infiltrate, peaks at 10 days and diminishes thereafter. The peak chronic inflammatory response reaches a level rated 4 at 20 days and remains constant from that point on. The chronic phase was composed of macrophages scavenging the dying fat cells, as well as a multinucleated giant cell response (foreign body). The most intriguing finding was the occurrence of mi-
+
crocysts that paralleled the loss of viable adipocytes. Their importance is that their number may reflect the long-term viability, with numerous cysts predicting ultimate graft death. CONCLUSIONS
Autografts of fat need to be studied more thoroughly in the laboratory before their universal acceptance as the “ideal” soft tissue substitute can be accepted. From
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our experiments, it appears that the model described in this article is a very useful and applicable animal model to study and evaluate fat autografts. These grafts follow a very predictable histologic pattern over time, as summarized in the article. Survival of some small portion of the fat graft does occur. This amount histologically appears to be in the range of 20% to 30%. Progressive fibrosis does occur in these autografts and may account for the final volumes obtained by some surgeons. Multinucleated giant cells, as well as macrophages, are the cells that scavenge the dying fat cells. The presence of microcysts predict ultimate graft death and resorption. In conclusion, liposuction (or suction-assisted lipectomy), is a commonly performed cosmetic surgical procedure. Autografts of fat obtained in this manner are being used for a variety of facial cosmetic and reconstructive procedures. In light of our histologic findings, the question of long-term graft resorption and fibrosis must be considered. REFERENCES 1. Rubin L. Biomaterials in reconstructive surgery. St. Louis:
CV Mosby Co, 1983. 2. Ellenbogen R. Free autogenous pearl fat grafts in the face-a preliminary report of a re-discovered technique. Ann Plast Surg 1986;1 6179. 3. Newman J, Levin J. Facial lipo-transplant surgery. Am J Cosmetic Surg 1987;4:131-40. 4. Asken S. Autologous fat transplantation: micro and macro techniques. Am J Cosmetic Surg 1987;4:111-21.
5. Bircoll M. Autologous fat tissue augmentation. Am J Cosmetic Surg 1987;4:141-9. 6. Agris J. Autologous fat transplantation: a 3-year study. Am J Cosmetic Surg 1987;4:95-102. 7. Neuber F. Fat grafting. Chir Kongr Verh Dtsch Ges Chir (in German) 1893;22:66. 8. Czerny M. Reconstruction of the breast with a lipoma. Chir Kongr Verh (in German) 1895;2:216. 9. Tuffier T. Treatment of pulmonary gangrene and abscesses. Troisieme Congres Societe Internationale Chirurgie (in French) 1911:780. 10. Zipper J. Fettransplantation. Beitr Klin Chir 1912;81:155. 11. Peer LA. Loss of weight and volume in human fat grafts. Plast Reconstr Surg 1950;5:217. 12. Illouz Y-G. Communications at the Societe Francaise de Chirurgie Esthetique. (June 1978 and 1979). 13. Bircoll M. Autologous fat transplantation. California Society of Plastic Surgery: March 1985. 14. Gurney CE. Experimental study of the behavior of free fat transplants. Surgery 1938;3:680. 15. Fischer G. Autologous fat implantation for breast augmentation. Workshop on liposuction and autologous fat reimplant. Isola d’Elba: September 1986. 16. Dolsky RL. Adipocyte survival. Presented at the Third Annual Scientific Meeting of the American Academy of Cosmetic Surgery and the American Society of Lipo-suction Surgery. Los Angeles, Calif., February 1987. 17. Johnson G. Body contouring by macroinjection of autogenous fat. Am J Cosmetic Surg 1987;4:103-9. 18. Breadon GE, Kern EB, Nee1 HB 111. Autografts of uncrushed and crushed bone and cartilage. Arch Otolaryngol 1979;105:7580.
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Injectionof autologous adipose tissue removed via liposuctionhas been used clinically for facial contouring, the aging face, furrows, facial atrophy, acne scars, nasolabial folds, chin, and various other surgical defects. Survival rates for autografts of fat have been quoted anywhere from 30% to 80%. Our study uses a reproducible rabbit animal model for autotransplantation of adipose tissue and examines the histopathologic changes that occur to the graft over time. Autogenous subcutaneous fat was removed from a dorsal scapular donor site, treated to stimulate cannula damage as in liposuction, then relnjected at the base of the ear. Histologic examination of the grafts were made at 5,10,15,20,40, and 100 days after transplantation. Hematoxylin-eosinsections were graded on degree of fibrosis present (0 to 4+), viable fat (1 to lo), degree of inflammation (0 to 4+), and neovascularization ( + or -1. Viability of fat decreased from 8.5 to 10 at 5 days to 2 viabillty at 40 days. Acute inflammation peaked at 10 days, followed by the chronic Inflammatory response with macrophages and multinucleated giant cells scavenging the dying fat graft. Neovascularization began at 5 days, peaked at 10 days, and remained constant thereafter only at the edge of the graft. Microcysts appeared at 15 days and Increased in number in proportion to the decrease in viable fat. In summary, the temporal histologic events are progressive fibrosis; decreased amount of viable fat; inflammation beginning with a neutrophillc response, later a macrophage and giant cell response; and neovascularization at the periphery of the grafl insufficient to maintain graft viability. In our animal model, autografts of fat appear to have limited long-term viability and are replaced by fibrous tissue. This may have clinical implications in autografting of fat in facial plastic and reconstructive Surgery. (OTOIARYNGOL HEAD NECK SURG 1990;102:314.)
I n the practice of facial plastic and reconstructive surgery, correction of soft tissue defects is a common and often difficult procedure. The perfect soft tissue substitute is yet to be discovered. Various synthetics, including paraffin, liquid silicone, encapsulated silicone, silicone rubber, plastic, and various collagen extracts have been tried. Recently autogenous transplantation of fat obtained via liposuction has gained favor among facial plastic surgeons. 2-6 Historically, the first use of free autogenous fat transplants was described by Neuber in 1893.7 He transplanted small pieces of upper arm fat to correct a depressed facial scar. In 1895, Czerny' used a large lipoma to fill a breast defect.8 Later, in 1911, Tuffier' described
From the Department of Otolaryngology, Mayo Clinic. Presented at the Annual Meeting of the American Academy of Otolaryngology-Head and Neck Surgery, New Orleans, La., Sept. 24-28, 1989. Submitted for publication Sept. 26, 1989; accepted Oct. 30, 1989. Reprint requests: Jeffrey Bartynski, MD, Department of Otolaryngology, Mayo Clinic, 200 1st St., NW, Rochester, MN 55905. 2311118434
pathologically the outcome of transplanted fat in the extrapleural space. The material was examined 4 months after transplant and found to be absorbed and replaced by fibrous tissue. In 1912, Zipper" examined microscopically free fat transplants after two mammoplasties and found encapsulated fat, as well as normal fat. Peer" performed a classic series of experiments in human beings in 1950. Thirteen cases of transplanted fat were moved in large blocks. Another thirteen subjects received the same weight of graft, but in smaller pieces. All fat was transplanted into the rectus abdominus sheath. The fat was later removed and examined histopathologically. The results revealed that neovascularization begins by the fourth day after transplantation and that there is some graft liquification. However, only about 45% of the original volume was lost in I year. He concluded that autotransplanted fat does indeed survive, refuting theories that histiocytes would absorb the fat and replace the graft. The next several decades saw the advent of synthetic implantable material gain widespread favor for implantation with its easy availability and lack of secondary donor-site problems. Then, in 1977, Illouz12introduced
314
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Histopathologic evaluation of adipose autografts 315
the technique known as liposuction. Initially used as a procedure to rid the patient of aesthetically unpleasing fatty deposits, it also provided the facial plastic and reconstructive surgeon with an abundant supply of potential graft material. In 1984, Bircoll13 first presented his technique of injecting autologous adipose tissue removed by liposuction. He described fat injection in facial contouring and for treatment of the aging face, facial furrows, midfacial atrophy, breast reconstruction, and cosmetic chest augmentation. Transplanted fat pearl autografts were resurrected by Ellenbogen* when he reported his experience with acne scars, nasolabial folds, chin, and eyelid depressions. In 1987, Newman and Levin3 summarized their experience with facial lipotransplant surgery, injecting such sites as melolabial folds, malar region and inframalar groove, forehead and glabella, and the mentum. The widespread clinical use of autologous fat obtained by liposuction for transplantation has progressed at a much faster pace than basic research. Actually, little basic science work has been done specifically looking at autologous fat transplantation. Survival rate for transplanted fat has been reported anywhere from 30%14 to as much as 80%.15 Methods of assessing adipose tissue viability are mostly performed on either a volumetric basis or from histologic study. ' 1 . 1 4 . 1 b . 1 7 Animal models have been used for various autografts and allografts. An excellent model to study implantable materials is the rabbit ear model of Breadon, Kern, and Neel. From their papers, histologic cross sections of the normal rabbit ear demonstrate no endogenous fat. Thus it would seem that the rabbit ear would be an ideal location to test autografts of fat. METHODS
A modification of the rabbit ear model used by Breadon, Kern, and Neel was used for this study." New Zealand white rabbits were each injected with autografts of fat at the base of each ear. Each rabbit was followed for a distinct predetermined period of time and cared for in accordance to the guidelines set forth by the institutional animal care committee. Anesthesia was induced with 35 mglkg of ketamine, mixed on a 9: 1 ratio with xylazine, then injected intramuscularly into the lateral thigh region. Each rabbit was given 600,000 units benzathine penicillin G intramuscularly in the hind leg before the procedure. A 5 x 5 cm paraspinal (interscapular) donor site was shaved, cleaned with thimerosal and 85% ethyl alcohol, and draped in a sterile manner. The area was infiltrated with 5 cc of I % lidocaine 1 : 100,000 with epinephrine (wet technique). The fat was harvested as follows: under sterile conditions a 1.5 cm skin incision overlying the fat pad was made with a #15 blade. The fat pad
was then sharply dissected with scissors and harvested. This fat packet was minced with scissors into a petri dish containing lactated Ringer's solution and cut into approximately 1-mm pieces. These pieces of fat were transferred into a 10-cc h e r lock syringe with control rings and passed through a 3.3-mm accelerator aspiration needle and cannula (Byron Medical, Tucson, Ariz.) to simulate cannula damage in liposuction. Fat to be injected was placed into a microinjection gun (THEGUN, Byron Medical, Tucson, Ariz.), which injects 0.1 cc with each click. An 18-gauge blunt-tipped needle with introducible trocar is attached to the tip. The rabbit ear recipient site on the dorsal (convex) surface was shaved, cleaned with thimerosal and 85% ethyl alcohol, and draped in a sterile fashion. The GUN, with 18-gauge blunt-tipped needle and trocar attached, was tunneled subcutaneously until the tip of the cannula rested 1 cm from the base of the ear. Fat obtained by the above method was split into equal sample volumes and injected into each ear, raising a bleb. Volume necessary to raise the bleb was recorded (usually 4 cc). The graft was observed for any signs of rejection (i.e., erythema, induration, and/or skin slough). The rabbits were killed by a lethal injection of pentobarbital at 5 days, 10 days, 15 days, 20 days, 40 days, and 100 days after transplant. The ears were removed and preserved in neutral buffered formalin. After the grafts were carefully dissected from their bed, serial sections were cut, imbedded in paraffin, and hematoxylin-eosin stains made. Representative sections were examined histologically under 20 x magnification. Ten random microscopic fields were examined and graded on degree of fibrosis (0 to 4 + ); viable fat (0 to 10); inflammation (0 to 4 + ); and neovascularization ( + or -). Predominant inflammatory cell type, multinucleated giant cell response, and microcysts were observed and their temporal relationship noted. RESULTS
Distinct histopathologic events occurred over time in the rabbit fat autografts. In the grafts examined at 5 days, the degree of fibrosis was very minimal and rated 1 . A very thin capsule was noted surrounding the graft and adherent skeletal muscle was noted. Viability of the fat appeared histologically to be very good, with normal fat architecture and normal adipocytes throughout the graft. Using our rating system, the viability at day 5 was rated anywhere between 8.5 and 10. Examination of the inflammatory response revealed a 1 + to 2 + response. The predominant inflammatory cell type seen was neutrophilic; a very limited number of macrophages were encountered. Most of this inflammatory response occurred at the periphery of the graft. Neovascularization was noted to occur in fibrous septa
+
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316 EvWTYNSKI et 01.
P
.
*
s,
d
L
i.
Fig. I . Day 5.Skeletal muscle bed with thin capsule surrounding normal adipose tissue [autograft).
Fig. 2. Day 5.Normal fat with neovascularization noted along graft edge.
near the periphery only, with the center of the graft remaining avascular (Figs. 1, 2, and 3). Ten days after transplant, the fibroblastic response was rated 2 + , with thickening of the surrounding capsule and increased fibrous septa. Histologic viable fat decreased to a 7 rating, with fat necrosis and collapse of the adipocyte walls encountered in the center of the graft. Acute inflammation peaks at 10 days at 3 + , with numerous neutrophils seen engulfing the adipocytes.
The beginning of the chronic inflammatory response is noted with macrophages accumulating, lipid near the graft edges. Few multinucleated giant cells are also first encountered at this time. Neovascularization peaks, but only at graft edges (Figs. 4, 5, and 6). The graft at 15 days shows little change from 5 days earlier, with a similar fibroblastic response and a viable fat rating of 6. An inflammation of 3 + is seen with an equal number of neutrophils and macrophages. The first
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Histopathologic evaluation of adipose autografts 317
Fig. 3. Day 5.High-power view of neovascularizationwith neutrophils in fibrous septa.
fl-
Fig. 4. Day 10. Capillary filled with RBCs along the graft edge.
appearance of microcysts is encountered at 15 days. The importance of these cysts will be noted in later specimens. Vascularization of the graft remains constant and only at the periphery. At 20 days, the grafts demonstrate a great deal of histologic change. Fibrosis increases dramatically, with a 3 + rating and a thick capsule and islands of fibrosis throughout. A marked drop occurs in the amount of histologic viable fat, to a rating of 4. Inflammation is
now almost entirely of the chronic cellular type and 4 . Lipid-laden macrophages are seen, as well as a rise in the number of microcysts-especially where viable fat is absent. The rise in microcysts parallels the decline in viable fat. Vascularity of the graft remains isolated to the margins of the graft (Figs. 7 and 8). Forty days after transplant, fibrosis reaches a maximum rating of 4+, with fibrosis throughout the substance of the graft. Viable fat is only rated 2, with few
+
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r”
t
Fig. 5. Day 10.Note increasing neutrophilic response on this view.
Fig. 6. Day 10.Area of exuberant neutrophilic infiltrate.
isolated “islands” of histologically viable fat. The chronic inflammatory response remains 4 + , with numerous macrophages scavenging the dying adipocytes. Also, a rise in the multinucleated giant cell (foreign body) response is noted at this time. Neovascularization remains insufficient and isolated. The graft itself is composed predominantly of microcysts and fibrous tissue when examination is performed at 40 days (Figs. 9 and 10).
Grafts examined at 100 days after transplant demonstrated little change from the histologic preparations made at 40 days. The progressive fibrosis and diminished fat viability had stabilized. A similar amount of microcysts were noted. SUMMARY The temporal histologic events occurring in autografts of fat obtained via liposuction in a rabbit model are as
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Histopathologic evaluation of adipose autografts 319
Fig. 7. Day 20. Multinucleated giant cell response noted on this view.
c
Fig. 8. Day 20. Areas of graft composed of predominantly microcysts.
follows. Graft fibrosis follows a progressive course, with an initial capsule formation followed by fibrous septationsand exuberant fibrosis of the entire graft. This fibrous reaction peaks at only 40 days after transplant and remains constant, with only slight progression noted at 100 days. Viability of the fat itself also diminishes over time. Initially on histologic evaluation, the grafts appear 80% to 100% viable. This diminishes gradually, however,
up until 15 days, and drops precipitously at 20 days to 40% viability histologically. The final viability appears to be 20% to 30%, as interpreted from our histologic preparations. Neovascularization begins along the graft periphery by 5 days. This neovascularity never proceeds past its peak at 10 days, and only occurs along the graft edges. Thus the graft never achieves an adequate blood supply at its center to maintain its long-term viability.
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Fig. 9. Day 40.Multinucieated giant cells scavenging a microcyst of fat with surrounding fibrosis.
Fig. 10. Day 40.Fibrous septa in center of the graft. Note the small vessel supplying an island of normal fat.
Inflammation was characterizedby acute and chronic phases. The acute phase, composed predominantly of neutrophilic infiltrate, peaks at 10 days and diminishes thereafter. The peak chronic inflammatory response reaches a level rated 4 at 20 days and remains constant from that point on. The chronic phase was composed of macrophages scavenging the dying fat cells, as well as a multinucleated giant cell response (foreign body). The most intriguing finding was the occurrence of mi-
+
crocysts that paralleled the loss of viable adipocytes. Their importance is that their number may reflect the long-term viability, with numerous cysts predicting ultimate graft death. CONCLUSIONS
Autografts of fat need to be studied more thoroughly in the laboratory before their universal acceptance as the “ideal” soft tissue substitute can be accepted. From
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Volume 102 Number A April 1990
Histopathologic evaluation of adipose autografts 321
our experiments, it appears that the model described in this article is a very useful and applicable animal model to study and evaluate fat autografts. These grafts follow a very predictable histologic pattern over time, as summarized in the article. Survival of some small portion of the fat graft does occur. This amount histologically appears to be in the range of 20% to 30%. Progressive fibrosis does occur in these autografts and may account for the final volumes obtained by some surgeons. Multinucleated giant cells, as well as macrophages, are the cells that scavenge the dying fat cells. The presence of microcysts predict ultimate graft death and resorption. In conclusion, liposuction (or suction-assisted lipectomy), is a commonly performed cosmetic surgical procedure. Autografts of fat obtained in this manner are being used for a variety of facial cosmetic and reconstructive procedures. In light of our histologic findings, the question of long-term graft resorption and fibrosis must be considered. REFERENCES 1. Rubin L. Biomaterials in reconstructive surgery. St. Louis:
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