Modern biological fillers

Currently, in the United States, biological materials derived from human or animal tissues are used more often than synthetic biomaterials. The most popular materials for soft tissue augmentation are autofat and bovine collagen. With the development of laboratory culturing techniques, the choice has expanded to include injections of a mixture of human collagen and cultured fibroblasts.

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Materials obtained from human autologous tissue

Autologous fat injection

The unpredictability of the results of using autologous fat grafts was realized quite quickly; it was associated mainly with local resorption of the transplanted fat. Two decades after Neuber's report on the successful transplantation of free fat grafts, Bruning first described the technique of fat injection. He placed small pieces of fat in a syringe and used it to correct deformities after rhinoplasty. In 1950, Peer indicated that the mass of freely transplanted fat decreases by an average of 45% after 1 year. He proposed a theory of cell survival, postulating that living fat is ischemized after collection, some fat cells die, and the tissue is resorbed and replaced by cystic structures and fibrous tissue. Other authors have shown that fat grafts harvested surgically retain volume better than those obtained by suction. With the introduction of liposuction, described by Illouz in the late 1970s, large volumes of fat tissue became available for implantation.

The microlipoinjection technique consists of fat harvesting, storage and reimplantation. Fat is harvested under aseptic conditions, local anesthesia, using the hypotonic infiltration technique, a blunt microcannula or syringe into a sterile container. The harvested fat can also be frozen in liquid nitrogen for future use. Potential donor sites include the lateral surfaces of the thighs, buttocks and abdomen. Serum and blood are separated from the fat, which is then washed with sterile saline. The transplanted fat is injected into the subcutaneous tissue with a wide perforating needle. After injection, the tissue is massaged to evenly distribute the injected fat. Indications for microlipoinjection include correction of nasolabial and buccal-labial folds, grooves on the bridge of the nose, lips, and hemifacial atrophy. Since resorption of the injected fat is expected, hypercorrection by 30-50% is recommended. In more mobile areas, absorption is accelerated, so repeat injections may be required to achieve long-term results.

In addition to donor-site complications, potential complications of microlipoinjection include mild edema and ecchymosis at the injection site, which usually resolve within 72 hours. There is one report of unilateral blindness following glabellar autofat injection. Resorption of fat grafts requires repeated injections, and replacement of the graft with fibrous tissue is a major concern with this technique.

Lipocyte correction of the dermis

In 1989, Fournier developed a modification of autologous fat injection. He proposed that if adipocytes were ruptured and the triglyceride content removed, the remaining cell walls and intercellular fibrous septa could be used as a connective tissue filler to correct dermal changes. He called this tissue autologous collagen, believing that it was rich in these fibers.

Coleman and colleagues reported excellent clinical results with good tolerability. The durability of this material was comparable to that of Zyplast (derived from bovine collagen) or Fibrel (porcine collagen), especially if the filling procedure was repeated after 2-4 weeks. Early biopsies revealed not intact adipocytes but significant inflammatory infiltration. Subsequent biopsies showed expansion of the dermis and replacement of inflammatory cells by cellular fibrosis. Interestingly, biopsies showed very low collagen content in the grafted material itself. Instead, the injection causes collagen deposition by the host.

Although this method is technically more difficult and time-consuming than using Zyplast or Fibrel, it appears to be safe and gives acceptable clinical results. It can be useful for the correction of perioral atrophy and skin scars. It can also be combined with other techniques such as microlipoinjection, laser treatment, or botulinum toxin A (Botox). The procedure often requires repetition. However, due to the abundance of donor material, it can be cost-effective, especially for large defects.

The fat is collected in the same way as for microlipoinjection. It is allowed to settle in the syringe for a few minutes so that the fat can be separated from the liquid part of the aspirate. The obtained fat is then collected in small syringes, with 1 cm3 of sterile distilled water added to each 2 cm3 of fat, and then frozen in liquid nitrogen. The syringes are then quickly defrosted in warm water. This results in separation of the supernatant from the fat residues, which are removed. The remaining triglycerides are separated from the injection material by centrifuging the syringes for 1 min at 1000 rpm. The treated tissue can be injected intradermally through a 23 G or 25 G needle.

The durability of the lipocytic correction material is similar to that of Zyplast collagen. Due to the expected absorption, some hypercorrection or repeated injections must be used. The side effects and complications of this procedure are the same as with microlipoinjection.

Autodermal transplantation

Dermal graft harvesting involves dissection, excision, and reimplantation of small, monolithic pieces of autodermis as a means of increasing soft tissue volume. Originally used in ophthalmology and voice restoration, dermal grafts are used to treat retracted scars, skin folds and wrinkles, and deep, wide soft tissue defects. They are not as effective in treating fine lines or small acne scars. Large acne scars, at least 4-5 cm in diameter, respond better to this treatment.

Scar undercutting as a single intervention has been shown to improve the appearance of these defects by separating the fixing fibrous bands from the skin, forming new collagen and fibrous tissue, and thereby lifting the defect area. However, after such an intervention, the undercut scars, in many cases, eventually reattach to the underlying fibrous tissue. The introduction of dermal grafts after undercutting can theoretically prevent reattachment to the fibrous tissue and provide longer-lasting results. Undercutting is first performed with an 18 G NoKor needle (Beckton-Dickinson, USA) in the midplane of the dermis. Viscous resistance is observed when cutting the fibrous bands. The introduction of dermal grafts is performed 2-6 weeks after the initial undercut.

The retroauricular area can be a good donor site. The advantages of this area are that it has a relatively low blood supply, contains few hair follicles and other skin appendages, and that the scar after tissue harvesting will be hidden. Under local anesthesia, the skin is dermabraded with a coarse diamond disc to the level of the deep dermis. The dermabrasion technique provides speed and precision in depth; however, the epidermis can also be removed with a scalpel or laser. The dermis can then be harvested with a scalpel to the level of the retroauricular fascia and immediately placed in cold sterile saline. The donor site is closed with absorbable sutures. The dermal graft with fat is divided into suitable fragments. Smaller grafts, 4-6 mm, can be used for smaller acne scars, while larger strips of dermis can be used to lift larger defects and to correct the sacral folds and nasolabial folds. The recipient sites are undercut with an 18 G NoKor needle at the middermal level and the graft is then inserted through the needle hole and positioned. For larger recipient sites, such as the nasolabial folds or lips, the graft can be pulled under the undercut area by a thread tied to one end. Both ends of the subcutaneous tunnel are then closed with fine absorbable sutures, incorporating the graft into the stitches.

Side effects and complications of this procedure include local bruising, hematoma, discoloration, swelling, crusting, and pain. Wound infection is uncommon but possible. Inclusion bodies in the form of epidermal cysts occur if the epidermis is not completely removed from the graft. Newly formed cysts may be painful but are treatable with incision and drainage. Graft displacement is rare and may require additional grafting. Scarring at the incision site may occur but is treatable with sanding.

Successful correction has been reported in 40-70% of cases after one procedure and 50-100% of cases after two procedures. Some shrinkage is observed for 1-6 months after treatment; therefore, some overcorrection is recommended when transplanting the graft.

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Cultured human fibroblasts (Isolagen)

With the introduction of new tissue culture techniques, it has become possible to easily grow large quantities of fibroblasts]. Fibroblasts from tissue cultures may serve as a potential dynamic, living filler for scar correction. Isolagen (manufacturer Isolagen Technologies, USA) is a product of such a process. The company processes autologous retroauricular skin biopsies and prepares syringes containing 1-1.5 cc of injectable autologous fibroblasts in a biocompatible medium over 4-6 weeks. Isolagen is injected into the superficial, middle, and deep dermis with a tuberculin syringe with a 30 G needle. To obtain 95% fibroblast viability, the material must be injected within 24 hours of delivery. Viability decreases to 85% and 65% after 48 hours and 72 hours, respectively. This technique is proposed for the correction of wrinkles, nasolabial folds, grooves above the bridge of the nose, scars, and hypoplastic lips. The recommended treatment consists of three to four injection sessions over a period of three to six months. In a study of over 100 patients with an 18- to 30-month follow-up, the rate of good and acceptable results was approximately 80%, with no significant complications or hypersensitivity reactions. Isolagen can be combined with other skin resurfacing procedures or bovine collagen injections to enhance the effect. Although the product and concept are promising, long-term results have not yet been studied. Isolagen is still being studied for approval by the US Food and Drug Administration.

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Injectable autocollagen (Autologen)

Autologen (manufacturer Collagenesis Inc, USA) consists of intact autologous collagen fibers obtained from autodermis dispersed in an injectable form. The collagen fibers are in the form of a suspension in a sterile phosphate buffer with a neutral pH. This product is usually offered in a standard 4% solution (Autologen) or a 6% preparation with cross-linked fibers (Autologen XL), but can also have a concentration ordered for a specific patient. Since the material is made from the patient's own skin, theoretically it should not cause allergic or immunological reactions and cannot be a carrier of diseases.

Autologen is indicated for the treatment of facial folds, dermal contour defects, and scars. It is injected into the mid-dermis with a 27-30 G needle. Injection to the desired depth causes a moderate blanching of the overlying skin. The suspension does not contain a local anesthetic, so the injection may be painful. A retrospective analysis of the procedure in 25 patients demonstrated correction of facial folds for up to 3 months after a single injection in 50-75% of cases and for up to 6 months in 50%. These results have not been replicated by other users.

The main disadvantage of Autologen is that the skin must be taken from the recipient. Previously, skin from blepharoplasty, facelift, brow lift, abdominoplasty, scar revision and other cosmetic surgeries has been used. Once the tissue is obtained, it can be frozen for up to 2 weeks or sent immediately to the company's lab. The yield is approximately 1 ml per 5 cm2. Limited histopathological studies have not revealed a significant inflammatory response to Autologen injections. Further clinical trials are needed to evaluate long-term results.

Materials derived from homologous human tissue

Acellular matrix of human skin (AlloDerm)

Soft tissue augmentation with homotissue has yielded variable results. Although autografts are generally preferred, their use is limited by donor site complications. AlloDerm (LifeCell Corp, USA) is an acellular dermal matrix derived from human allodermal skin obtained from tissue banks in the USA. The graft is freeze-dried without damaging the intercellular matrix and maintains the structural and biochemical integrity of the dermis and basement membrane. Cells are removed by dissociation of their matrix bonds and changes in calcium concentrations, ionic strength, and acidity, combined with the use of low molecular weight buffered detergents. This process produces a nonimmunogenic graft. It remains stable when frozen for 2 years and rehydrates with 10-20 min of soaking in saline or lactated Ringer's solution immediately prior to use.

In clinical settings, AlloDerm was first used to treat deep burns. Since then, it has been used in a variety of cosmetic and reconstructive surgeries as a soft tissue augmentation material. Experience with AlloDerm for lip augmentation, correction of nasolabial and buccal-labial folds, and folds above the bridge of the nose has been satisfactory. The powdered product has also been used as an implant to smooth out retracted scars.

To correct nasolabial folds, a 3 x 7 cm piece of AlloDerm is cut diagonally into two triangles. Each segment, after rehydration, is twisted along the long axis. Some surgeons fix the free edges of the graft with absorbable sutures. However, in such situations, a minimal amount of suture material should be used to prevent inflammation. Incisions are made at the corners of the lip in the groove at the base of the nostrils; a subcutaneous tunnel is created with an elevator. The graft is then pulled through it and gently massaged to correctly position the graft along the tunnel. The incisions are sutured. Antibiotics are prescribed locally and orally for several days.

Atrophic perioral folds can be corrected by lip augmentation using AlloDerm. Typically a 3 x 7 cm piece of graft is used, which after rehydration is folded as described above. Excess graft can be trimmed to achieve the desired shape and thickness. The use of sutures to maintain the shape of the graft should be limited as much as possible. Some surgeons place the graft without any sutures, allowing it to take the shape of a tunnel. Small incisions are made in the oral mucosa, slightly lateral to the oral commissures, and a submucosal tunnel is created bluntly just below the vermilion border of the lip. Care must be taken not to damage the orbicularis oris muscle. The graft is then inserted into the tunnel and evenly distributed by massaging the lip. Excess material is trimmed and the incisions are sutured. Maximum swelling in the area of the graft occurs 3 days after surgery. Local and systemic antibiotics are administered for several days. Patients with a history of viral infection are recommended to take prophylactic acyclovir.

Hypersensitivity and infections requiring graft removal are rare. There is a report of 30-50% volume loss at the surgical site after 1 year. There are also two reports of 65-70% retention of grafts after 18 months in mobile sites and 100% retention of volume in fixed sites after 2 years. These early reports of durability have generally been refuted by clinical practice. Most surgeons using this material report retention for 6-18 months.

Homologous injectable collagen (Dermalogen)

Dermalogen (manufacturer Collagenesis Inc, USA) is an injectable human collagen fiber suspension prepared under aseptic conditions from human donor tissue obtained from tissue banks in the United States. The drug is regulated by the Food and Drug Administration as a human tissue graft. Like Autologen, Dermalogen suspension is acellular, preserving intact collagen fibers. Donor material is collected and intensively processed to prevent the possibility of transferring infectious agents. Indications and methods of application of Dermalogen are identical to those of Autologen, but without the need for autologen skin sampling. It is available in 3%, 4%, and 5% concentrations and is injected with a 30 G needle. The product comes with a skin test portion, which should be done 72 hours prior to surgery. Preliminary data indicate preservation of the shape of the correction area for 6-12 weeks. However, data on the long-term results of using Dermalogen have not yet been obtained.

Materials not obtained from humans

Injectable Bovine Collagen

In 1977, Kparr et al. were the first to report the development of a highly purified collagen preparation from bovine dermis. They administered purified human and bovine collagen to 42 patients for 20 months. The preparation was purified, filtered, and dialyzed in phosphate-buffered saline with 0.5% lidocaine. In all cases, preliminary testing was performed by injecting 0.1 ml of material. Complications included cellulitis, pemphigus, and skin hyperpigmentation. No differences were found between human and bovine collagen.

As a result of these efforts, in 1981 the injectable collagen Zyderm I Collagen Implant (manufacturer Collagen Corp, USA) received approval for marketing from the Food and Drug Administration. It became the first non-autologous soft tissue augmentation product legally approved in the United States. Zyderm Collagen is made from bovine skin and is a purified suspension of collagen obtained from bovine dermis. Its production involves purification, enzymatic digestion and sterilization. After purification, bovine collagen is digested with pepsin and reconstituted in saline containing 0.3% lidocaine to a final concentration of 35 mg/ml. This process removes most of the antigenic determinants from the telopeptide regions of the molecule without disrupting the natural helical structure, making bovine collagen more compatible with human tissue. The intact and almost non-specific helical portion of the product consists of 95-98% type I collagen and 2-5% type III collagen.

The main limitation of Zyderm is the short-term nature of the corrective effect due to rapid absorption of the implant. To overcome this problem, more concentrated preparations, Zyderm II and Zyplast, were introduced. Zyplast is collagen cross-linked with glutaraldehyde, which increases its lifespan and reduces antigenicity. Unlike Zyderm I and Zyderm II, Zyplast is designed primarily for deep dermal injection. In a comparative study of Zyplast and Zyderm, Kligman and Armstrong found Zyplast to be more stable. However, it causes a greater inflammatory response with fibroblastic infiltration and collagen deposition, whereas the bulk of Zyderm is absorbed after 3 months without a fibroblastic reaction. Even Zyplast, despite its relatively greater durability, requires repeated injections.

Patients are tested by subcutaneous injection of 0.1 ml of material into the palmar forearm. The injection site is examined after 48-72 hours and after 1 month. Development of redness, induration, or both after 6 hours constitutes a positive test result and contraindication to the use of the implant. A 3-3.5% rate of positive skin tests and 1.3-6.2% false negative results have been reported. Therefore, a repeat test after 2-4 weeks is recommended. Therapeutic injections can be started 2-4 weeks after a negative result of the repeat skin test.

Adverse reactions associated with collagen injection include transient erythema, edema, ecchymosis, local skin necrosis, local granulomatous reaction, and abscess formation. Mild transient erythema may be expected shortly after injection. The incidence of skin flap necrosis secondary to compromised arterial supply following deep dermal injections of Zyplast has been shown to be 0.09%. Abscess formation is associated with hypersensitivity. Painful, tense cysts are treated with incision and drainage. This complication is rare (4:10,000) and may persist for more than 2 years. In several studies, circulating antibodies to Zyderm have been identified in some patients following single or multiple injections. These circulating antibodies to bovine collagen have been found to cross-react with human collagen. Sensitization may be related to either previous injections or to beef consumption. People with the HLA antigen DR4 may be genetically more susceptible to hypersensitivity.

Gelatin based implant

Spangler reported the first use of fibrin foam as an injectable filler for facial scars and folds in the 1950s. Fibrin foam was a fractionated plasma product containing fibrinogen and prothrombin. When injected under retracted scars, fibrin deposition, fibroblast infiltration, and new collagen formation occurred. Gottlieb took this concept further by developing a technique using gelatin, aminocaproic acid, and plasma. Gelatin served to elevate the retracted scar and promote clot formation; aminocaproic acid stabilized fibrin by inhibiting fibrinolysis, and the patient's plasma supplied clotting factors. The gelatin-based implant Fibrel (Mentor Corp, USA), an evolution of this technique, was approved for sale by the Food and Drug Administration in 1987. It is sold as a kit consisting of a lyophilized mixture of 100 mg of absorbable gelatin powder and 125 mg of e-aminocaproic acid. Porcine gelatin is used, so a skin test is required, which is assessed after one month. Some patients with allergic reactions to Zyderm do not develop hypersensitivity to Fibrel. Fibrel has gained acceptance mainly for intradermal injection for the correction of retracted scars. Significantly fibrosed scars do not respond well to Fibrel. Fine lines on the eyelids, lips, and wrinkles from photodamage also do not respond well to Fibrel injections due to the viscosity of the implant and the inflammatory response.

A study of scar and fold correction in 321 patients over 5 years showed that the result was maintained in 80% of cases after 2 years and in 50% of cases after 5 years. Adverse reactions included local erythema, swelling, itching, bruising, and pain. No serious systemic adverse reactions were observed when Fibrel was administered to 288 patients. Although Fibrel has been reported to be less allergenic and immunogenic than bovine collagen, its use requires more time for preparation and plasma collection. Combined with greater discomfort than collagen injections, these factors have limited the use of Fibrel.

Hylan B Gel (Hylaform)

The biocompatibility of the hyaluronic acid molecule, combined with its insolubility in water and resistance to degradation and migration, has made this substance an attractive means of increasing the volume of the dermis.

The molecule has no species or tissue specificity, as the chemical structure of this polysaccharide is the same in all animal species. Hylaform (manufacturer Biomatrix Inc., USA) is a purified preparation of animal-derived hyaluronic acid obtained from cockscomb. This preparation is used to treat retracted skin scars, folds and wrinkles. Hylaform gel is produced in a concentration of 5.5%. A series of injections is usually required for maximum correction. Complications include temporary local erythema, hematoma and nodule formation. Hylaform is available in Europe, Canada and other countries, but has not yet been approved by the Food and Drug Administration.

Restylane

Restylane (manufacturer Q-Med, Sweden) is a third-generation cross-linked, stabilized, non-animal hyaluronic acid gel with a high molecular weight (20 mg/ml), obtained from bacteria by fermentation. It is sold in sterile syringes containing 0.7 ml of material. The drug is injected into the dermis to a medium depth with 27 G or 30 G needles. In Europe, it is used to correct folds above the bridge of the nose, nasolabial folds, oral adhesions, retracted post-acne scars, and for lip augmentation. Up to 50-80% of the correction volume is preserved after 6 months. The main complications are erythema and edema at the injection site. Like Hylaform, this drug is absorbed over time. Restylane is not sold in the United States.

Resoplast

Resoplast (manufacturer "Rofil Medical International BV", Netherlands) is the first injectable collagen implant manufactured in Europe. Composed of monomolecular bovine collagen, Resoplast is available in 3.5% and 6.5% solutions and requires skin testing. Indications, injection technique and results are similar to those of Zyderm or Zyplast. This product is currently not available in the United States.