Introduction

Elastin confers on tissue the ability to stretch and recoil and plays a critical role in supporting and maintaining healthy tissue. In the skin, most elastin is located in the dermis. Generally, the more mature, thicker elastin fibers are found deeper in the dermis, where they function as an interpenetrating elastin network.

The building blocks of elastin therapy

The protein tropoelastin is the fundamental building component of all elastin. The production of tropoelastin mainly occurs before birth and in the first few years of life when the elastin required for the body to develop is produced. In adults, the low level of elastin production may mean that damage cannot be efficiently repaired and the skin gradually loses its elasticity.

The primary transcript from the single ELN gene is spliced to give different forms of the tropoelastin protein that either lack or contain various exons, which in turn give rise to forms of tropoelastin that may vary slightly in their protein sequence. The implications of this splicing are not clear, although some exons remain while others are occasionally spliced out. For example, exon 26A is unique to humans and appears to be spliced out in healthy elastic skin tissue but may be present under conditions of elastin damage, such as following UV exposure or extreme temperature treatments, as reported by Schmelzer et al. Therefore some forms of the tropoelastin protein may be associated with healthy elastic tissues and other forms with injury or disease.

As a key step in making elastin, many tropoelastin molecules associate and are then cross-linked, or connected, to form insoluble elastin. The process of elastic fiber formation is also known to include a number of other molecules. Microfibrils, of which fibrillin-1 is the major component, are structures present in the extracellular matrix and are thought to anchor elastic fiber formation. Cross-linking tropoelastin spherules are introduced to the microfibrils by the molecules fibulin-5 and possibly fibulin-4 and accrete on pre-existing elastin. Chapman et al found that fibulin-2 may work cooperatively with fibulin-5 to assist in elastic fiber formation. Emilin-1 may also regulate oxytalan fiber formation but, in a study by Nakatomi et al, did not appear to directly regulate elastin expression or deposition. The cross-linking of tropoelastin is carried out by lysyl oxidases – a family of five enzymes (LOX and LOXL, LOXL 2–4) that are likely to redundantly contribute to the cross-linking process. Maki et al found that LOX knockout mice show a reduction in elastin cross-linking. In addition, in studies by Noblesse et al, both LOX and LOXL were detected by immunohistochemistry in the dermis and epidermis of normal human foreskin and dermal equivalents with expression levels shown to decrease with age.

The raisons d’être of elastin regeneration and injecting elastin products

Damaged elastic fibers are responsible for an aged and wrinkled appearance (Fig. 9.1). Given the importance of elastin to the skin and its loss in the aging process, it is not surprising that various attempts have been made to maintain or replenish elastin levels. Treatments aiming to repair or regenerate elastin in elastic tissues should consider all the molecules implicated in elastin fiber formation. However, as elastin fibers develop, they ultimately consist of over 90% elastin and so the integration of sufficient tropoelastin into elastin fibers is clearly the major target. Effective treatment approaches are restricted owing to the obvious physical challenge of transferring materials and / or treatments across the epidermis and into the dermis, resulting in a preference for small molecules and physical treatments. Tretinoin or all-trans retinoic acid is a small molecule utilized for many years in topical formulations to increase elastin production through increased tropoelastin and fibrillin expression and secretion. Molecules such as aldosterone and mineralocorticoid receptor antagonists may impact on elastin fiber deposition in skin. Soy and rice (USPTO 19469891) extracts may also increase elastin formation, as can a combination of zinc and copper. More recently, a dill extract has also been shown by Cenizo et al to have the potential to promote elastin formation by promoting LOXL synthesis and secretion into the dermis.

Figure 9.1 Wrinkled skin due to loss of elastic fibers in a patient aged 12 years. Example of a male with acquired cutis laxa. Six months after presenting with an inflammatory condition, the patient developed generalized cutis laxa with marked sagging and wrinkling of the skin.

Courtesy of Hu Q, Reymond JL, Pinel N, et al 2006 Inflammatory destruction of elastic fibers in acquired cutis laxa is associated with missense alleles in the elastin and fibulin-5 genes. Journal of Investigative Dermatology 126(2):283-290.

However, the major challenge to overcome is the very low level of expression of tropoelastin in adult skin, as such treatments are likely to have only incremental benefits on the density of skin elastin, as reported by Sephel et al.

A new approach to treat aged and damaged skin using elastin is currently in clinical development in Australia by Elastagen Pty Ltd. The approach is unique to skin augmentation treatments in that it is based on a recombinant human tropoelastin protein, which is identical to the one naturally occurring in healthy human skin. As seen in early clinical studies, this high degree of similarity to the natural elastin protein promises a significantly higher level of tissue compatibility than achieved with animal-derived products or synthetic polymer materials (Fig. 9.2). The treatment process, trademarked as elastatherapy^®, also benefits from a new formulation chemistry that enables the tropoelastin protein to be cross-linked with a low-concentration HA component. There is no requirement for the toxic cross-linking agents often used in other products and the use of hyaluronidase to correct poor treatment outcomes is still an option – a significant advantage over alternative dermal filler materials. Elastin treatments benefit from the cell-supporting properties of elastin and the potential to stimulate skin cells, leading to the formation of new collagen at the treatment site. This latter property coupled with the product’s smooth, cohesive structure may present significant advantages over particulate products, which target collagen regeneration but carry a significant risk of lumps and nodules. The product range also includes the potential to bulk the skin with a long-lasting, highly biocompatible elastin material and the unique prospect of restoring elastin to improve the skin’s physical properties and suppleness.

Figure 9.2 Hematoxylin and eosin staining of upper arm dermis following biopsy 7 days after implantation. Note the irregular bands of the brightly eosinophilic fibrous elastin deposit amongst the dermal collagen in the reticular dermis.

Courtesy of Dr Robert Daniels, Elastagen Pty Ltd.

Ongoing clinical development will help identify the unique potential of what will be the first recombinant human elastin product as an aesthetic skin treatment.

Introduction

Platelet-rich plasma (PRP) is an autologous concentration of human platelets in a small volume of plasma. Multiple alternative terms to PRP are used in the literature including autologous platelet gel, plasma-rich growth factors, and autologous platelet concentrate. The use of growth factors permeates many fields of medicine and surgery, including facial rejuvenation and plastic surgery, maxillofacial surgery, dentistry and oral surgery, tissue engineering and research and development, cardiovascular surgery, orthopedic surgery and sports medicine, gastroenterology, and urology.

Concentrated platelets are a rich source of seven fundamental protein growth factors actively secreted by platelets during the initiation of wound healing. Human growth factors have been investigated extensively and clinical applications of individual growth factors such as keratinocyte growth factor and platelet-derived growth factors are used for the treatment of oral mucositis and non-healing diabetic wounds, respectively, as reported by Scalafan.

The growth factors present in platelets comprise three platelet-derived growth factors, two transforming growth factors (TGF1 and TGF2), vascular endothelial growth factor, and epithelial growth factor. PRP also contains fibrin, fibronectin, and vitronectin – known to act as cell adhesion molecules for osteoconduction and as a matrix for bone, connective tissue, and epithelial migration.

The raisons d’être for the use of platelet-rich plasma in cosmetic and reconstructive medicine

Method

PRP production has been greatly simplified so that it can be used in the office and operating room. A sterile centrifugation process allows platelet separation from red blood cells and their sequestration in high concentrations without lysing the platelets or damaging them so that they no longer can actively secrete their growth factors. Many ‘kits’ are available and the resultant separation is illustrated in Figure 9.3, in which the plasma utilized from the separated elements is the yellow supernatant.

Figure 9.3 Separation after centrifugation into blood elements with the straw- or yellow-colored supernatant being the platelet-rich plasma component.

In essence, preparation is now relatively simple and quick and may be completed in physicians’ rooms.

Pearl 5

Current semiautomated methods allow ‘in house’ preparation of platelet-rich plasma in a clinical environment.

Prior to treatment a simple blood sample of 8–20 mL is extracted from the patient. This blood is immediately placed in the centrifuge for 8 minutes. Centrifugation separates plasma from erythrocytes. The platelet-rich plasma is combined with calcium chloride and injected into the skin.

Evidence

Despite the myriad of articles in the area of platelet use in dermatology and plastic surgery, and indeed in many other disciplines, the variation in preparation protocols, kits, activation methods, platelet concentrations, and growth factors still leaves many questions unanswered. The same frustration is seen in sports medicine where injection therapy is commonplace but lack of consensus has led to differences in application methods, timing of treatment, and volume of injection. Appropriately powered level 1 and 2 studies with adequate and relevant outcome measures and clinically appropriate follow-up in order to assess the efficacy and effectiveness of all elements of PRP therapy has been called for by Mei-Dan et al. A review of the literature would suggest a similar need is present in cosmetic medicine.

Platelet preparations have applications in surgery and are especially useful for the soft tissue and bony reconstructions encountered in facial plastic and reconstructive surgery. Their use in these situations has been associated with a decrease in operative time, necessity for drains and pressure dressings, and incidence of complications.

Platelet-rich plasma has been applied to wounds, providing hemostasis, adhesion, and enhanced wound healing. A potential recent use for improving wound healing after ablative fractional CO₂ laser resurfacing has been reported by Lee et al.

Pearl 6

Platelets may have a role as both a topical agent and injected into skin and other tissues for repair and rejuvenation.

The dermal and subdermal placement of blood products has quite a long history, with subcision a potent technique thought to rely on physical disruption of scar attachments but also on the wound healing and clotting cascade of the wound-healing process (as reported by Orentreich & Orentreich 1995) and delayed or incomplete fibrinolysis has been seen as a mechanism of dermal augmentation with Fibrel^®. Whole blood (unclotted) was injected into scars in the study by Goodman 2001, but it appears that platelets not erythrocytes are the most important in repair post-injury and at promoting cytokine release, as reported by Jacobson et al 2008.

There appears to be a jostling for commercial advantage with different systems promoting different methods of proprietary PRP formation and indeed different methods of using this PRP.

There have been relatively few manuscripts dealing with injection of these platelet gels intra- and subdermally (see the articles by Sclafani and by Redaelli et al) and no controlled studies have so far been performed. In a study of 15 patients with moderate to severe nasolabial folds, the Wrinkle Assessment Score (WAS) prior to treatment was 3.30 with a decrease in the score by 0.65 at 1 week increasing to a difference of 0.97, 1.08, and 1.13 at 2, 6, and 12 weeks, respectively. The small sample size still allowed statistical significance to be achieved at the 2-, 6-, and 12-week observations. The method used in this paper was observation for a relatively short time and was only a single injection, whereas other guidelines have recommended two or three injection sessions. It may be that sustained release of growth factors occurs.

Recently, histological changes associated with PRFM) injection into the dermis and subdermis of human skin have been examined by Sclafani & McCormick. These investigators found that PRFM treatment led to dermal neocollagenesis within 7 days of treatment. By 19 days, treated areas additionally showed angioneogenesis and adipogenesis. By the conclusion of the study at 10 weeks, these changes were still present but fibroblasts, endothelial cells, and adipocytes appeared mature and stable.

Conditions that preclude the use of PRFM include: aspirin ingestion, vitamin E-containing medications, warfarin therapy, sepsis, acute and chronic infections, fibrinogen disorders, very low platelet counts, and any coagulation disorders.

Case Study 1

(Courtesy Dr A. Sclafani,)

A 67-year-old female with Fitzpatrick grade 2 skin requested facial rejuvenation, particularly around her eyes. Physical examination revealed significant involutional and gravitational changes in her mid-face, with skeletonization of her infraorbital rims and nasojugal troughs. The patient was treated with autologous PRFM, with 2.75, 2.25, and 1.00 mL injected bilaterally in a supraperiosteal plane during three visits over a 6-month period (the last treatment was performed 4 months after the second to accommodate the patient’s travel plans). Each treatment was terminated when there was slight overcorrection. The patient reported a roughly 25% loss of correction over the first week after treatment after each treatment, but the degree of correction was relatively stable after the first 2 weeks. The patient has been pleased with her results, which have been stable over the past 9 months.

Case Study 2

Figure 9.4 illustrates the treatment of a patient with severe hollows below her eyes using autologous PRFM.

Figure 9.4 Patient with severe hollows below her eyes: (A) Prior to treatment with autologous platelet-rich fibrin matrix (PRFM); (B) 1 month after first treatment with PRFM; (C) 3 months after third treatment with PRFM.

Introduction

A dermal filler, Laresse^® (Fziomed, San Luis Obispo, CA), composed of carboxymethyl cellulose (CMC) and polyethylene oxide (PEO), is described for soft tissue augmentation. Laresse^® was designed using measurements of the viscoelastic and physical properties of marketed dermal fillers composed of cross-linked HA. In a pilot study, 12 patients with moderate or severe nasolabial folds (NLFs) were randomized to contralateral treatment of similar volumes of Laresse^® or Restylane^®. Both Laresse^® and Restylane^® were effective in providing clinical correction for up to 6 months. Related adverse events were similar for both products and typical of dermal filler products.

The study indicated that Laresse^® was similar to Restylane^® in safety and effectiveness, providing NLF correction for up to 6 months. Subsequently additional patients were treated with NLF injections and / or lip augmentation with Laresse^® without complications other than expected mild edema of 1–2 days’ duration.

Non-cross-linked PEO/CMC fillers such as Laresse^® can be formulated to have rheological properties similar to cross-linked HAs. They behave as a viscoelastic gel with dynamic and rotational properties that are similar to cross-linked HAs (Falcone & Berg, in preparation). The CMC/PEO-based Laresse^® described here had a CE mark, but for commercial reasons is not presently available.

Characteristics

Laresse^® is composed of CMC and PEO at a concentration of 35 mg/mL in physiological saline and calcium chloride. The filler has the appearance of a viscoelastic gel and is easily injected through a 30-gauge needle. Restylane^® is composed of cross-linked particles of HA. Laresse^® is a solution of two pharmaceutical-grade polymers that are not cross-linked. The two gels are similar in terms of the percentage elasticity, indicating that they have similar apparent stiffness (Fziomed data on file).

Pilot clinical study

An observer- and patient-blinded study over 6 months was performed in the UK with recruited subjects seeking soft tissue augmentation for correction of bilateral NLFs. A single treatment session was randomized so that treatment of NLFs occurred with Laresse^® on one side and Restylane^® on the contralateral side, followed by an evaluator-blinded 6-month follow-up. Following initial screening, including a forearm skin test with Laresse^®, each patient received Laresse^® or Restylane^® on contralateral sides of the face after perioral anesthetic nerve injection with Xylocaine^® 1% allowing intrapatient comparison of treatment outcomes. Both the patient and evaluating investigator were unaware of treatment allocation. Injection technique of both fillers was similar, i.e. tunnelling with a 30-gauge needle followed by filler injection on retraction of the needle. However, treatment allocation could not be concealed from the treating investigator owing to the different packaging the two fillers.

The response to the initial injection of Laresse^® or Restylane^® was evaluated after 2 weeks for adverse events. Patients were further evaluated at 1 month, 3 months, and 6 months post-treatment. As Laresse^® had limited clinical use prior to this study, subjects participating in the study were required to have a skin test 28 days prior to treatment. This was negative prior to treatment in all patients. This was not our practice in post-study treated patients.

Photographs for baseline Wrinkle Severity Rating Scale (WSRS) scoring were taken of each patient using standardized photography. Each side of the patient’s face was randomized with respect to treatment procedure. Follow-up visits were at 1 month, 3 months, and 6 months post-treatment.

A masked, trained evaluator determined WSRS scoring using a five-point scale. In addition to the masked, trained evaluator, the patient and the treating investigator determined scores using the Global Aesthetic Improvement Scale (GAIS). These were conducted at 1, 3, and 6 months after the treatment session. Day et al made efficacy comparisons between study treatments on the mean change from baseline in the WSRS score of the NLFs as determined by the masked evaluator at all follow-up timepoints. The WSRS has demonstrated its robustness in previous clinical trials (by Narins et al and Day et al) of intradermal fillers. Scoring wrinkle severity is based on visual comparison of the length and apparent depth of the NLFs against an agreed set of reference photographs of NLFs. The WSRS is a five-point scale with values of 1 (absent), 2 (mild), 3 (moderate), 4 (severe) or 5 (extreme).

In addition, the overall change in appearance of the NLF from its pre-treatment condition was determined at each follow-up visit during the double-blind phase using the GAIS reported by Narins et al. The GAIS is a relative five-point scale with values of 1 (worse), 2 (no change), 3 (improved), 4 (much improved), or 5 (very much improved) from pre-treatment. Both the patient and the treating investigator scored the patient using the GAIS. An archival (pre-treatment) photograph was kept for each patient and this was used as the reference image at each follow-up visit.

Case Study 3 Clinical results

Laresse^® study

All 12 patients completed 6 months’ follow-up. The average volume of gel implanted in either NLF was 1.81 mL of Laresse^® and 1.75 mL Restylane^®. The results from the study indicated that Laresse^® was effective in providing a clinical correction for up to 6 months, as was Restylane^®.

The WSRS scores for all 12 patients are plotted in Figure 9.5. The number of patients was too small to determine the level of statistical significance for a comparison of the two products. The GAIS scores were also determined from the responses of both the patient and the treating physician and results from the patients and the treating investigator indicate that the correction is still noticeable by the patient and the investigator at 3 and 6 months for both treatments. The mean baseline score for both groups was 3.4 and both fillers demonstrated measurable clinical correction at 6 months.

Figure 9.5 Comparative Wrinkle Severity Rating Scale (WSRS) scores of Laresse^® and Restylane^®.

The safety profile of both products was similar with adverse events consisting of swelling and firmness at the injection site.

One patient experienced a localized reaction of redness and itching within 2 minutes on the side treated with Laresse^®. This type of reaction has been observed in the past with Restylane^® by Andre et al. The localized redness was treated with ice and topical hydrocortisone and improved within 2 hours. It completely resolved within 23–48 hours, the patient completing the study and maintaining clinical correction for 6 months. Based on similarity to other cross-linked HA, Laresse^® was considered to be safe. Induration involving the injection site affected most of the patients for both groups and generally resolved within 1 week. This induration is presumed to be a result of local trauma of injection resulting in localized edema and inflammation. Importantly there were no delayed-onset local reactions during the study.

The similarity in safety and effectiveness to HA filler suggested that Laresse^® is a promising, novel dermal filler.

Case Study 4

Figures 9.6 and 9.7 illustrate a patient treated with 1.8 mL Laresse^® to the nasolabial fold and upper lip.

Figure 9.6 A patient before (A) and after (B) 1.8 mL Laresse^® to the nasolabial fold and upper lip.

Figure 9.7 A patient before (A) and after (B) Laresse^® to the nasolabial fold and upper lip.