Hair Transplantation in Cicatricial Alopecia: The Role of Autologous Fat Transfer

Aim: The purpose of this article is to prove the importance of autologous fat transfer (AFT) in scarring


Background
In order to give a realistic introduction to this article, a brief digression is necessary: if a patient with cicatricial alopecia, (scalp, beard, eyebrows, etc.) wanted nowadays to find out which reconstructive options were available, his search would be difficult. Assuming he finds a few specialists with the highest expertise, he would most likely have to compare very different treatment plans. The answers he would receive are an honest reflection of the different backgrounds of the specialists involved in this field. Among them are doctors of different specialties: wigs specialists, a professional category that is often consulted by these patients as first choice; scalp micropigmentation specialists (SMP) who offer a rapid disguise solution that, especially for men, is very common nowadays (Table 1).

Introduction
For many years the relative advantages of autologous scar transplantation, compared to skin expansion, was the subject of controversy because the reliability of the two procedures appeared to be different [1]. Moreover, only a few surgeons were able to perform these procedures correctly and, in many cases, the transplanted hair grew back but differed from expectations in number and quality. Today, hair transplantation (HT) is the primary indication for the coverage of the majority of cicatricial alopecia. The previous limits of its applicability have been extended and should be redefined. In the last 4 years the increase of all HT operations worldwide has been exponential and ISHRS statistics show that 2% are performed on scars (ISHRS Survey 2018) [2]. How is it possible to achieve similar results to those achievable in non-scarring alopecia? When the greatest obstacle is represented by the qualitative impairment of the receiving tissue, the adoption of regenerative medicine plans represents a real and sustainable solution to recreate the right suitability of the scar tissue for surgery.
We present this first clinical work for three main reasons: i. Describe how the AFT technique, Microfat (Micro) and Lipostructure (Lips), is able to induce an improvement in the quality of scarring recipient areas. ii.
Specify the reasons, indications and scientific backgrounds behind the adoption of this choice. iii.
Present our clinical experience highlighting both the wide variety of indications for the use of adipose tissue, as well as the patient selection criteria, surgery plans and various technical notes.
If the exact regenerative mechanism by which the stem component of adipose tissue acts remains unclear, we have observed that the proposed approach produces a formidable effect on the follicular structure. In the field of hair care and surgery the potential of these regenerative procedures is tremendous, and few first report are available [3,4]. It is rare, as it is shown in (Table 2), for these skills to flow into a single subject, and these patients can only be managed by open professional cooperation. This work, which stems from these synergies aims to promote this multidisciplinary approach.

Materials and Methods
In the last three years we have observed 30 patients with scarring alopecia of the scalp or facial areas. Of these 30, 21 have completed the procedures and are now at a minimum distance of 6 months from the date of HT. 9 patients are still undergoing treatment and have been excluded from this study. Patients enrolled for this procedure plan presented one or more clinical evidence of reduced wound thickness, impaired tissue trophism, altered circulatory function and structural abnormalities with particular reference to adherence to the deep planes. Possible comorbidities were investigated through dermatoscopy and treated before the procedures were performed. Moreover, to accurately define the worth of HTs, all patient in this study underwent to a coverage value (CV), a routine investigation we have identified as an important tool to meet this purpose. The CV is a method to measure the total donor capacity (TDC) of a patient during the preliminary assessment. TDC is the maximum number of FUs that a surgeon can harvest (respecting the hairs/FU ratio) in each procedure while avoiding depletion. It is based on an arithmetical calculation using quantitative (number of FU/cm 2 e number of hair/FU) and qualitative (hair caliber) data. The CV is the ratio of Hair/cm 2 multiplied by the Average Caliber of the FU. A CV of 5, 4 is the value that represents the lightest coverage necessary to reach a good aesthetic result (Table 3). So far, we can determine how many grafts will be required in the recipient area in order to allow a minimum coverage or a specific coverage value [4,5]. This density will come from the grafts placed and a precise calculation of the real recipient area (which can often differ from the perceived recipient area) [5].

COVERAGE VALUE FORMULA
This data will also help to choose if HT will cover completely or partially the scar, using the available resources to transplant exclusively the areas of primary importance from an aesthetic perspective. More importantly, within this specific use on unhealthy tissue, it highlights what useful reserve of follicles the patient has in case of partial success of the procedure. The CV analysis takes about 30 minutes. Recently two apps have been proposed in order to increase the speed and the accuracy of this procedure [6,7]. An example of CV calculation is shown in (Figure   1). Of the 21 patients examined for the purpose of this study, 19 had lesions which originated from secondary causes while 2 had lesions which originated from primary pathologies (GVHD and Romberg scleroderma. The latter two cases have been included because they have been stable for more than three years. All patients underwent a reconstructive sequence consisting of one or two AFT interventions and then one HT. Table 4 provides a summary of the operations performed.

I Autologous Fat Transfer (AFT)
Two different AFT techniques, Microfat and Lipostructure, have been performed. The choice falls on the preference of the surgeons and the need for a greater or lesser volumetric increase.

i Microfat a Preparation
The microfat procedure is performed under local anaesthesia. The fat collection is made using 1mm diameter microperforated and microuncinate cannula with 0.8mm holes, significantly smaller than traditional ones (SEFFIHAIR® produced by SEFFILINE S.r.l. Bologna -Italy). The tunnelings are performed in the subcutaneous tissue adjacent to the dermis; this layer has been proven to hold more mesenchymal and vascular stem cells [8,9]. Once the adipose tissue is harvested, it is transferred into 1ml luer lock syringes. The details of this technique are described in a previous article [10].

b Technical Notes
Under ring block anaesthesia of the receiving area, the suspension is injected using 21 or 23G needles into the subcutaneous layer at different levels. A fan pattern is followed using a retrograde injection technique to reduce thromboembolic risks. Some scarring lesions of the scalp have an extremely reduced thickness (1-2 mm) and in these cases, in order to overcome the limited thickness, we performed a preliminary infiltration of the receiving area with 1-2cc of PRP in order to expand the tissue promoting a multi-layer placement of the cellular fraction. The use of needles as an infiltration tool is necessary for two reasons. The first is to be referred to the frailty of some scar tissue, that is hard to penetrate with blunt micro-cannulas without running the risk of lacerations that could compromise the whole procedure. The second concerns the structural stiffness of some lesions, that may appear as a single block attached to the deep planes. In the latter, as other authors have highlighted, the cannula would not allow the positioning of the microfat in multiple layers [11].

ii Lipostructure
The lipostructure procedure is performed under local, local assisted or general anaesthesia using Dr. Coleman's technique [12,13]. In the most penetrable tissues a cannula has been used for fat positioning, in other tissues needles have been used for the same reasons described above. AFT interventions have been spaced from a minimum of 3 months to a maximum of 10 months. The greater the amount of fat grafted, the longer the waiting time for a second session.

II Hair Transplantation (HT)
Both Strip and FUE HT techniques were used in the cases presented [14,15]. The following are some technical peculiarities relevant to the procedures performed after the grafting of adipose fractions.

Technical Notes
HTs on previously grafted scarring lesions must consider four factors that differentiate them substantially from hair transplantations on healthy tissue. 1) The intense ischaemic response to vasoconstrictor use.
2) The lower graft retention by the tissue.
3) The greater frailty of the skin. 4) The greater vulnerability to infection. i.
Under normal circumstances, adipose tissue can withstand prolonged ischaemia, but recent experimental studies show that under stress conditions it is able to release inflammatory molecules [16]. At subsequent reperfusions, these can cause arteriolar vasoconstriction resulting in stagnation and blockage of capillary flow. These secondary phenomena can compromise the yield of the FU [17]. Hence the need not to use or severely restrict the use of vasoconstrictors in the receiving area by limiting them to peripheral use. ii.
AFT procedures can restore and regenerate the thickness and texture of the scar tissue by reducing the need to exaggerate the inclination of the follicles during positioning. The drawback is the reduction in the graft-retention-capacity of the tissue, due to the decreased number of elastic fibres. This can cause 'popping' during placement. The best prevention is to perform multiple preliminary tests to define the exact proportion between the incisions and the available grafts. For this purpose, it is important to have a wide range of blades or implanters of various sizes available. In addition, it is preferable to use the "stick and place" technique for the placement of 'oversize' grafts (single or wide three).
iii. The skin, while improving in quality with the adipose graft, loses stability due to a looser connection to the deep structures; the resulting abnormal mobility on the underlying planes may make it more prone to lacerations during the graft's placement. The manoeuvre must be adapted accordingly. To improve the visibility of the operating field, multiple sprays of saline solution are preferable to gauze padding. iv.
The increased vulnerability to infection, due to the absence of skin appendages, requires the adoption of sterility measures for this operation that are usually not required in normal HT. All patients are required to start antibiotic therapy beforehand. A short video summary of the method described is available online (Link) [18].

Results
The results of the surgical plans carried out were documented photographically and dermatoscopically. In many cases a video recording was also performed to allow a better understanding of the density obtained in a dynamic situation. The photos and videos were submitted by the authors and two colleagues with experience in hair restoration surgery. definitive result in a single session. The authors' overall assessment is shown in (Table 5). The feedback from the patients was largely positive for everyone and for those who had already experienced challenging surgical experiences due to underlying pathologies. It should be mentioned that even in the case of electrocution, where a superficial infection occurred, the patient stated that they had very little discomfort and showed excellent compliance. Three results are shown in (Figures 2-4). The most noticeable difference from the numerous cases of HT performed in the past years on scarring lesions was the constant and early hair regrowth that started between the second and third month after the transplant. It is unusual to observe the first results of the procedure after only four months ( Figures 5 & 6). This phenomenon is to be considered in relation to a synergy of circumstances and positive actions and has often been the premise of excellent and stable results. The contrast with the past, characterized by interventions without preliminary regenerative operations, has represented for the authors a perfect litmus test based on which it is possible to state here that the benefits of these procedures are clear and evident.

Discussion
Recent technical improvements, inherent to HT's two main procedures, have mainly focused on a myriad of changes aimed at perfecting a "secure transfer" of FUs from the donor site to the recipient site. The new procedures have reduced all kind of damages to the grafts during both the harvesting and the placement phases. Moreover, improved storage solutions improved their homeostasis during the ischaemiareperfusion period. These changes have had a significant impact on the quality and reliability of the results and have undoubtedly contributed to the explosive demand for HT in the aesthetic field. Minor attention has been given to the receiving area. Mesotherapy, PRP and regenerative medicine (ADSCs) have been used in a very diversified way and mainly to consolidate the results of medical therapy and to stabilize the results after HT. This retrospective study comes from the observation of increasing clinical evidence of complete and early regrowth of follicular units after HT in AFT pre-treated scarring areas. The selection process when approaching patients for this scientific work, was based solely on a well-structured clinical evaluation. There is, therefore, a lack of instrumental evaluation of results in quantitative (counting) and qualitative (hair thickness) terms. The authors reserve the right to investigate these aspects further in a future study. These evaluations are based on the authors' expert clinical judgement, dermatoscopy and global video photography data.
Why replace a single operation with a multi-step sequence? Which considerations about past experiences have led to this point? We know that several types of resident cells are needed for a tissue to be functional, able to maintain homeostasis and to perform its regenerative functions. So far enriching an impoverished tissue seems intuitively an advantage, but this has proven to be not enough. Equally, it could be said that hair transplantation was already an ideal tool for this purpose. [19,20]. The hair follicle already enriched the tissues with all its components and one single HT could achieve the same goal. In fact, the positive effects of HTs alone in cicatricial alopecia has been known for years and both HT (strip and FUE) have been widely use. Strip technique was predominantly used for these cases until 2015, giving also the option of maintaining a habitual hair style [21]. Moreover, grafts derived from a Strip preserved a greater amount of adipose perifollicular tissue, they were less skeletonized, offering the chance to implant a true composite graft.
It was clear that FUs richer in adipose tissue required more nourishment to ensure their survival particularly in damaged scar tissue. These limitations ended in a tendency to perform conservative transplants at low density. In order to be more predictable. the reconstructive path was frequently divided into several steps of HT's. Beyond the common sense that derives from experience, the surgeon had no tools to determine the right number of grafts to use in a previously compromise area. The unsatisfactory yield of the FUs would have led to a frustrating result, worsening the clinical picture due to a significant waste of hair follicles. Given that the FU availability is not only exhaustible but often limited. In this context, patients were frequently disappointed with the partial results, many of which could have led them to abandon the reconstructive path. The refinement of the FUE technique has offered many new options. Among them is worth mentioning the PAHT (peripheral areas HT), a recent proposal by Dr. Sebastián Yriart to extend the harvesting area to the regions surrounding the scar considering the AGA evolution scenario [22]. Even considering all these new strategies, the necessity to improve the trophism of the receiving tissue prior to perform HT to overcome the reliability of the results remains the same [23].
The reasons for the need to change course are resumable in two main points: i. Improve the reproducibility and consistency of results. ii.
Significantly reducing the risks of the procedures and improving their course of action.
Significant advantages have been obtained when comparing this approach to interventions carried out "tout court" and not combined with preliminary steps. A previous treatment of the scar tissue means that it can be transplanted with greater density and reduces need for multiple sessions of HT. Not only this procedure plans accelerate the achievement of the final result, but frequently a satisfactory result for the patient can be seen from the first HT and reduces the risks of unsuccessful operation.
The surgeon's work is more predictable and more options to create this "safe starting point", prior to an HT are available: 1) Amount of fat to be transplanted.
3) Time-intervals between each session. 4) Standardized protocol to harvest and manipulate the micro fragmented adipose tissue.
i. The amount of AFT used in each single treatment can be proportional to the characteristics of the area to be treated and it can be progressively increased. ii.
The number of sessions, in our clinical experience, is limited to two but can be adapted to each case. iii.
Intervals between session must be chosen to maximize the final result. iv.
In 50% of the cases presented, the preparation of the autologous fat was carried out using a single standardised protocol (SEFFI). This system, created with the purpose of being used in a similar way by surgeons with different backgrounds and skills, always offers the advantage of performing the harvesting with the same instrumentation and guidance. The adipose tissue fragments are aspirated from the same subcutaneous layer and do not require any substantial manipulation prior to grafting (Figure 7). This has created a standard that allows for a better results comparison and which we intend to investigate further in the future.
The Procedure Manual aspiration/selection of the adipose tissue.  A further advantage is the long-term durability of the HT. This factor, frequently underestimated, is of crucial importance. This study has in fact shown how this technique promotes a longer and increasingly stable lifespan of the FUs in HT's. The immunomodulatory action provided by ADSCs and SVF fragments could allow a greater predictability of longterm results also of HTs in primary scarring lesions (Lichen, AFF, etc.) that today are strongly conditioned by recrudescence of the same pathology. The two cases included in this study have been monitored for more than three years and present a stable clinical picture.
Despite all these considerations, a reflection to be considered in the therapeutic proposal, as far as the density to be achieved is concerned, comes from the analysis of cases of women who have developed chemotherapy-induced alopecia (CIA). A picture of widespread alopecia that is not very responsive to treatment and that leads to a thinning of various degrees. These data provide precise elements to decide whether to operate or not by suggesting alternative techniques to patients with particular reference to prosthetic ones. For many of these patients, the density of a prosthetic is necessary to restore a satisfactory image. This experience leads us today to judge with greater criticality surgical results amongst females with previous scarring alopecia, whose expectations on density are measured on a different scale when compared to male patients. Managing expectations has to be part of a comprehensive patient discussion that needs to be crystal clear prior to approaching a surgical path, which can often only reach a partially satisfactory result.

Conclusion
This work further widens the field of application of the AFT which, like it already happened in maxillo-facial, plastic and cosmetic surgery, proves to be successful to transform complex procedures, associated with higher risks, into simpler procedures [24][25][26][27]. It aimed to explore how tissue regeneration through AFT can significantly improve hair growth in the area of scarring alopecia, underlining, once again, the tremendous potential of this approach. Since surgeons have been debating the causes responsible for reduced yield or loss of results in the long term, this article opens two big questions: 1) Will combining the preliminary clinical evaluation with instrumental evaluation redefine the concept of eligibility of a scar tissue to HT? 2) Will it be useful to extend the benefits of this method from scarring cases to those with only aesthetic purposes that, especially having already undergone a previous HT, are not free of these problems?
New strategies are being studied today to improve the results and survival of fat grafting. We believe that HTs and their results, easy to judge by their clinical evidence, could be a useful tool in the future to evaluate the real effectiveness of these improvements in clinical practice. This year we celebrate 500 years since Raphael's death, Italian renaissance painter and architect, who inspired the metaphor which we would like to conclude this article. The genius looking at a rough block of marble perceives the presence of a wonderful shape inside it. The