The aim of this study is to assess the efficacy of tSVF-enriched fat grafting in patients with adherent scars. The primary outcome measure is the change in scar pliability as assessed with the Cutometer Skin Elasticity Meter Dual MPA 580 (Courage and Khazaka GmbH, Cologne, Germany) pre- and 12 months post tSVF-enriched grafting. The secondary outcome measures are scar erythema and melanin index, as a proxy for color and pigmentation, measured by the DSM II Colormeter (Cortex Technology, Hadsund, Denmark), scar quality assessed by the patient and observer scales of the Patient and Observer Scar Assessment Scale (POSAS 2.0), and histological features of tSVF and scar biopsies.
Protocol and registration
This study was approved by the Dutch Central Committee for Clinical Research (CCMO) NL72094.000.20, and by the Institutional Review Boards of the participating hospitals (Red Cross Hospital, Beverwijk, and Martini Hospital, Groningen, the Netherlands). Methods of the study are specified in a protocol that is registered at the Dutch trial register (March 16th, 2020, NL 8461)38.
A prospective intra-patient before-after cohort-study on the effect of tSVF-enriched fat grafting on scar pliability in patients with adherent scars and who are treated in the Red Cross Hospital, Beverwijk, or the Martini Hospital, Groningen, the Netherlands (Figure 1).
Patients, aged ≥18 years with adherent scars (≥12 months old) caused by burns, necrotic fasciitis or degloving injury, visiting the outpatient clinic of one of the Burn Centers in Beverwijk or Groningen who have an indication for fat grafting are eligible for this trial. Exclusion criteria are: previous scar treatment with fat grafting, skin melanoma in patients’ history, pregnant or lactating, BMI <18 (general exclusion criteria for fat grafting procedure), unlikely to comply with the requirements of the study protocol and follow-up, and insufficient knowledge of the Dutch language (Table 1). Patients are included after they received a confirmed understandable and neutral explanation of the study by a member of the research team and after signing informed consent following the guidelines of the CCMO.
- Age ≥18 years
- Patient has an adherent scar (minimum scar age: 12 months) caused by burns, necrotic fasciitis or degloving injury, for which fat grafting is indicated
- Competent adults
- Patients seen by a plastic surgeon in Burn centers of the Red Cross Hospital, Beverwijk or Martini Hospital, Groningen, The Netherlands
- Previous scar treatment with fat grafting in selected scar
- General exclusion criteria for fat grafting procedure: pregnancy, lactating, BMI < 18 kg/m2
- Skin melanoma in patient’s history
- Unwillingness to commit to the study protocol and show up for all follow up moments
- Insufficient proficiency in Dutch to the extent that clear communication is not possible
Before surgery, one scar area will be selected and marked according to a standardized algorithm for objective data collection (Figure 2)31. The maximum size of the scar area is limited by the maximum quantity of tSVF that can be obtained and a minimum length of 2 cm will apply. Each scar consists of five measurement points. The five points in the selected area will be subjected to all study outcomes.
Under general anesthesia, lipoaspirate is harvested from the abdominal wall, flank or thigh with fine harvesting cannulas. Out of 10 ml lipoaspirate, 1 ml of tSVF will be produced intraoperatively with the Fractionation of Adipose Tissue (FAT) procedure, a fat dissociation procedure using a fractionator (Figure 3 and 4)39. For preparing the tSVF-enriched fat graft, 10 ml lipoaspirate in a 10 ml Luer-Lock syringe will be centrifuged at 3,000 rpm with a 9.5 cm radius fixed angle rotor for 2.5 minutes (Medilite, Thermo Fisher Scientific, Waltham, MA) at room temperature (Figure 5). Thereafter, the upper oily layer will be drained from the top while the bottom layer will be drained by removing the lower cap of the syringe. Subsequently in the syringe 9.0 ml of fat graft will be left to which is added 1.0 ml of tSVF: two syringes, one with 9 ml of fat and one with 1 ml of tSVF will be connected to a Luer-Lock and the contents will be mixed by gently pushing de content of one syringe into the other, this way yielding the tSVF-enriched fat. Small incisions at the border of the scar are created to perform adhesiolysis of the selected scar. Then, the tSVF-enriched fat (tenfold excess fat over tSVF) will be injected under the scar. This procedure is repeated until the entire scar area has reached fat grafting saturation (when enriched fat graft starts coming out of the incision holes). After fat saturation, the scar is covered and fixed with gausses. Practically, 50 ml of fat has to be harvested to yield 25 ml of fat and 2.5 ml tSVF, resulting in 27.5 ml tSVF enriched fat graft.
Primary outcome measure
The primary outcome measure is the change in scar pliability as measured by the Cutometer Skin Elasticity Meter Dual MPA 580 ® (Courage and Khazaka GmbH, Cologne, Germany) pre- and 12 months post tSVF-enriched fat grafting (Figure 2 and 6). The Cutometer ® is a validated, reliable instrument which measures viscoelasticity of the skin by analyzing its maximal extension after inducing negative pressure35,36.
Secondary outcome measures
All scar outcome measurements will be performed by two trained observers who work independently of each other to prevent confirmation bias.
Scar, retraction, elasticity, viscoelasticity and maximum extension will be measured with the Cutometer Skin Elasticity Meter Dual MPA 580 ® (Courage and Khazaka GmbH, Cologne, Germany) pre- tSVF-enriched fat grafting and 3- and 12 months post tSVF-enriched grafting (Figure 2 and 6)31. Scar pliability will also be measured after 3 months post tSVF-enriched fat grafting.
Healthy skin measurements will be conducted as described pre-, 3, and 12 months post-tSVF grafting on the contralateral side of the treated scar. Healthy skin measurements will serve as a reference (i.e. the optimum skin mechanics within that person).
Scar color and pigmentation
Scar color and pigmentation will be measured with the DSM II Colormeter ® (Cortex Technology, Hadsund, Denmark), a validated and reliable tool that calculates an erythema and melanin index as a proxy for color and pigmentation (Figure 2 and 7). Measurements of the selected scar and adjacent normal skin will be performed pre-, 3- and 12-months post-tSVF-enriched grafting31.
Scar quality will be assessed by the POSAS version 2.029,30. The POSAS Patient and Observer scales are completed pre- 3 and 12 months post-tSVF-enriched grafting (Figure 2 and 8). All items of the POSAS 2.0 will be scored on a 10-point rating scale and added together to obtain a final score. Additionally, item specific scores and the overall opinion of the scar will be scored (Figure 8).
A standardized photograph will be taken from each scar within 24 hours before treatment under the same light and camera conditions each time.
For participants in our study, scar biopsies are optional. If patients consent to biopsies, histological features will be assessed by taking a 2 mm biopsy of the selected scar on a fixed position; 1 cm left from point 1 within 24 hours before treatment and at 12 months post tSVF-enriched grafting 1 cm right from point 1 (Figure 2). Skin sample will be fixed with 2% PFA and paraffin embedded. From each patient, 0.5 ml of tSVF will be 2% paraformaldehyde (PFA) fixed. Fixed samples will be transported to the University and Medical Center Groningen for histological analysis. There, thin sections of paraffin embedded scar biopsies and tSVF will be deparaffinized and stained with H&E, Picrosirius red (extracellular matrix collagen), CD68 (macrophage infiltration), perilipin A antibodies (presence of residual adipocytes), alpha-smooth muscle actin antibodies (smooth muscle cells and myofibroblasts), and von Willebrand Factor and/or CD31 (endothelial cells). Stained sections will be scanned and subjected to image densitometry for quantification.
Baseline patient and scar characteristics
Patients’ baseline characteristics will be extracted from patients’ medical records and consist of the following: age, sex, skin type (Fitzpatrick), scar characteristics e.g. scar age, cause of the scar, prior surgery of the selected scar other than fat grafting (exclusion criterium), e.g. split skin grafting.
Sample size calculation
The primary outcome is the difference in pliability measured by the Cutometer (parameter Ue) between pre- and 12 months post--tSVF-enriched fat grafting. The expected effect size is based on the results of the study by Jaspers et al. on the Coleman fat grafting technique31. In this study, scar pliability improved by 22.5%. The mean pliability measure was 0.51. At least this effect is expected to be found in tSVF-enriched fat grafting as well. An additional positive effect of 20% of tSVF-enriched fat grafting (compared to the Coleman technique) is considered a clinically relevant improvement of treatment in this study. Therefore, a sample size calculation was made with an expected effect size of 0.45. With a required minimum of 80% power, a two tailed test and p<0,05 considered statistically significant the required sample size is 41 patients (G*Power 3 version 18.104.22.168). With an anticipated maximum dropout rate of 10% at 12 months, we will need to include 46 patients to compensate for drop-out.
Comparisons will be made for all primary and secondary outcome metrics of the selected scar and adjacent normal skin across all follow up moments, thus pre-, 3- and 12-months post tSVF-enriched grafting, according to the established data collection protocol31. Analyses will be performed using SPSS Statistics, version 27.0 (IBM Corp., Armonk, NY) and Image J (NIH, Behesda, ML) for histological examination of skin biopsies. If data is normally distributed, results will be presented as mean with standard deviation and the primary outcome will be analyses with the paired T-test. Furthermore, the repeated measures ANOVA will be performed to detect any overall differences between related means across all time points. If the repeated measures ANOVA is statistically significant, we will run appropriate post hoc tests to highlight exactly where differences occur. If data is not normally distributed, appropriate non-parametric analyses are performed. Data will be analyzed using SPSS and for histological analysis, Graphpad Prism (version 8.4; GraphPad Software, Inc., La Jolla, United States) will be used. P-values <0.05 will be considered statistically significant.