Addition of platelet concentrate to dermo-epidermal skin graft in deep burn trauma reduces scarring and need for revision surgeries

Abstract

Background: [corrected] Deep skin burn injuries, especially those on the face, hands, feet, genitalia and perineum represent significant therapeutic challenges. Autologous dermo-epidermal skin grafts (DESG) have become standard of care for treating deep burns. Additionally, human autologous thrombin activated autologous platelet concentrate (APC) has gained acceptance in the setting of wounds. While each of these interventions has been independently shown to accelerate healing, the combination of the two has never been evaluated. We hypothesized that the addition of platelets (source of growth factors and inhibitors necessary for tissue repair) to the DESG (source of progenitor cells and of tissue proteases necessary for spatial and temporal control of growth regulators released from platelets) would create the optimal environment for the reciprocal interaction of cells within the healing tissues.

Methods: We used clinical examination (digital photography), standardised scales for evaluating pain and scarring, in combination with blood perfusion (laser Doppler imaging), as well as molecular and laboratory analyses.

Results: We show for the first time that the combination of APC and DESG leads to earlier relief of pain, and decreased use of analgesics, antipruritics and orthotic devices. Most importantly, this treatment is associated with earlier discharges from hospital and significant cost savings.

Conclusions: Our findings indicate that DESG engraftment is facilitated by the local addition of platelets and by systemic thrombocytosis. This local interaction leads to the physiological revascularization at 1-3 months. We observed significant elevation of circulating platelets in early stages of engraftment (1-7 days), which normalized over the subsequent 7 and 90 days.

Trial registration: ClinicalTrials.gov NCT01383187.

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

The authors state that there are no conflicts of interest regarding the publication of this article.

Figures

Fig. 1

Post operative pain assessment after DESG. A) Visual Analog Pain Scale (VAPS): Patients treated with APC and DESG were evaluated using a VAPS from 0–10. As evident, most were pain free by day 6. The shaded area in A indicates historical experience with pain disappearance. B) Percentage of Patients on analgesics. Prior to surgery, approximately 78% of patients required analgesics, by the 2nd post-operative day, the number dropped to 56%, by the 4th post operative to 33% remaining on analgesics, and by 14th post-operative day (the end of the monitoring period) only 6% of patients require analgesics. C) Impact of Leukocytes on the Pain Evaluated in Visual Analog Pain Scale. The curves are gnostic distribution functions of leukocytes conditioned by a certain level of pain. Each individual curve represents the relationship between VAPS and leukocytes at a given time. Prior to DESG the VASPS some patients had 2/10 pain (black curve), or less than 2/10 pain(red curve), at 7 days, there are patients without pain (purple) and with pain over 1 (blue), by the 14th day only 6% of patients have pain (brown) over 1 and the rest is pain free (green). D) Interpretation of VAPS using Distribution Functions. The curves represent the conditional probability (in this case probability of c-reactive protein influencing pain). The diversion of the probability curves indicates that 82% of the patients with high CRP also had high pain prior to procedure. At 7 days those with pain and those without pain had nearly the same concentration of CRP, and finally at 14 days those in whom inflammation persisted had high pain (brown) and those with low CRP had no pain (green).

Fig. 2

Post operative pruritus assessment after DESG. A) Visual Analog Pruritus Scale (VAPrS): The range of the scale is from 0–10. As evident, degree of pruritus was lower than in patients not treated with DESG/APC (shaded area). Pruritus drops from pre-procedural levels, reaching a nadir at 7 days, and then raising again with healing (B). B) Percentage of Patients using antihistamines. Percentage of patients on antihistamines was used as a surrogate for pruritus and scar formation. On the 2nd and 4th post-operative day 17/18 patients (94%), and on 14th postoperative 14/18 (78%) patients did not require any antihistamines. C) Interpretation of VAPrS and eosinophils using Distribution Functions. Each individual curve represents the relationship between eosinophils and pruritus at a given time. Prior to surgery there was ~55% of patients who had low levels of eosinophils and high pruritus (note the value at which blue and black curves cross) and the remainder had low eosinophils and high pruritus (blue curve). At 7 days the low pruritus line (purple) and no pruritus lines (red) intersect at about 50%. Decrease of pruritus was correlated to absence of scarring, and the decrease of pruritus is in agreement with the finding that majority of patients continued not to need antihistamines throughout the observation period (12 months). D) Relationship between platelets and eosinophiles analyzed using Distribution Functions. Their inter-relationships reveal that at 7 days there are low eosinophiles and high platelets (significant at 0·1 eosinophiles, red line). At 14 days, platelets decrease, but remain higher with a peak of 600×109/L, and by 3 months both eosinophiles and platelets are in normal ranges. Most notably, the patients with pruritus had low levels of eosionophils throughout the study, suggesting that the mechanism of pruritus may not be histamine related. This would agree with the finding that those patients using anti-histamines to control pruritus do not get relief from the medication.

Fig. 3

Rate of Healing. The rate of wound healing was evaluated on a scale 0–100%. The majority of graft area (more than 99%) was healed in 33% of patients on the 4th post-operative day, in 61% on the 6th day, in 67 % on the 8th postoperative day, in 72% on the 10th day, 78% on the 12th day and 94% of patients on the 18th post-operative day. The healing seen with DESG and platelets occurs earlier is more robust.

Fig. 4

Vancouver Scar Score Scale. The scale score is based on: pigmentation (0–3), vascularity (0–3), pliability (0–5), and scar height (0–3) with a maximum achievable score of 14. The Vancouver Scar Score ratings of patients not treated with DESG and platelets range between 7–10 (grey shaded area), mainly because 40–60% of these patients have scar hypertrophy (1). In our study, patients treated with the combination show a remarkable decrease of scores with maximum scores achieved at 3 month (median=4) and high quality scar (median=2) from 12 months onward.

Fig. 5

Inflammation during re-epithelialization. A) Rate of post-operative re-epithelialization. The rate of epithelization was evaluated using a scale from none=3 points, early=2, sustained=1, and full=0. We found that most grafts (72% of patients) were completely epithelialized by 10th day. B) Conditional distribution functions correlating epithelialization and c-reactive protein (CRP) at 0, 7 and 14 days. It appears that better epithelialization on the 7th and 14th post operative day are related to a lower CRP (red and blue line). C) Distribution functions correlating epithelization and leukocyte levels. At 7 days all patients without healing have higher leukocyte counts (purple line) than those that healed (blue line. In contrast at 14 days the patients who have not healed have lower levels of leukocytes, while higher levels of leukocytes are correlated to better healing (red line). However, because the majority of patients (78%) were healed at 14 days, leaving few patients for calculation of this curve, it is difficult to reach the later conclusion. D) Dependence of epithelialization on the fibrinogen levels. Following healing (at 14 days) fibrinogen levels appear to have dropped (red line) to less than baseline (black line at day 0). At 7 days there appear to be only small differences between healers and non-healers.

Fig. 6

Distribution of individual coagulation parameters. A) Evolution of Distribution Functions of aPTT: While values of aPTT are higher than normal at 7 days (black curve vs red curve), they gradually normalize at 14 (blue) and 90 (purple) days. B) Evolution of Distribution Functions of TT. The values of TT are higher than normal at 7 days in about half of cases (black vs red curve), they remain elevated at 14 days (blue), and gradually normalize by 90 days (purple). The value of TT following the surgery (cross of the black line with blue and red) remains high and very variable in over 50% patients at 7 and 14 days, but normalizes at 3 months. C) Evolution of Distribution Functions of INR. The value of INR is elevated and very variable in nearly 80% patients at 7 days. At 14 and 90 days the values somewhat normalize, but never reach initial levels. D) Evolution of Distribution Functions of Platelets. Platelet levels at 7 days following surgery are significantly elevated and very variable in over 90% patients (cross of the black line with red), and they gradually normalize and correct to values less than those at initial (black curve) by three months.

Fig. 7

Evolution of distribution functions of hematological elements. Following DESG/APC grafting. There is evidence of initial blood loss (left shift of the red curve) in hemoglobin, hematocrit and red blood cells analysis, with gradual stabililization of the curves over time on the level substantially exceeding the initial state. Dynamics of changes in neutrophile, monocyte and lymphocyte levels is less considerable but not negligible.

Fig. 8

Correlation diagrams for cytokines interactions. Heat map diagrams of correlation coefficients of individual selected cytokines, where blue represents a negative correlation [−1 to 1] and red represents a positive one [1 to −1]. The color and size of each individual square indicates a quantitative measure of the correlation. The diagram shows the correlations of neutrophiles, platelets or MPV (always in the left lower corner of the diagram), with 3 cytokines (VEGF, SDF1, PDGF). In the case of neutrophiles (A) and platelets (B), the strongest positive correlation is between VEGF and neutrophiles (or VEGF and platelets in B). Mean platelet volume (B) has a very strong negative correlation with SDF1. The correlation of VEGF and PDGF is very weak in all three correlations.

Fig. 9

Histology of DESG/APC at the time of application. Hematoxylin/Eosin: 40x; Immunohistochemistry for bFGF, Platelet Derived Growth Factor (PDGF), SDF1, CD34; was performed on the graft tissues at the time of DESG/APC application. The expression of bFGF localized to the stroma of the germinal layer (blue arrowheads in C,D), and to the junction of keratin layer and epidermis. The expression of PDGF is localized in the stroma of the germinal layer (black arrowheads in E,F), an area commonly inhabited by progenitor cells. Dark red arrows in E,F point to CD34+ cells marked by red stain. The expression of SDF1 is localized to the stroma of the germinal layer (brown arrowheads in G,H), and fibroblastic cells (dark grey arrowheads in G, H). Dark red arrows in G,H point to CD34+ cells marked by red stain.

Fig. 10

Laser doppler imaging studies. Were donne on day 0, 6, 14 and 30. Day 0 is prior to any procedure. Each of the images has a defined region of interest (ROI) marked by a white border and labeled “1”. In patient 1 (A1–A5) a pre-operative scan, was done on the 4th post-traumatic day, and showed very low perfusion (46.4 perfusion units, PU), surrounded by inflammatory hallo (A1 on Fig. 10). On the 2nd post-operative day perfusion increased to 176 PU (A2). Next two scans indicate decrease in perfusion to 94 PU on day 4 and to 58 PU on day 8 before a secondary increase in perfusion that occurred with healing, and is evident by a rise to 167 at 93 days. In patient 2 (B1–5) the preoperative scan was acquired 12th post-traumatic day (day -2 of surgery) and showed low perfusion 94·9 PU, raising to 279·2 PU in the first 2 days post-op, before gradually decreasing to a nadir of 39·3 on the 8th post-operative day, and stabilized between 57·6–118·1 without a secondary peak. In patient 3 (C1–C5) was first scanned 7 days post trauma (day -1), and showed 81·5 PU, raising to 235·4 PU, dropping gradually to 37·2 on the 6th post-operative day, and stabilizing between 34·2 and 132 PU.

Fig. 11. Laser doppler perfusion study

A) Dynamic summary of perfusion studies are obtained by estimating the distribution functions of the 11 data sets of RPU (Relative Perfusion Units) for each of the 11 time points. Nine values of percentiles of each of these distributions are calculated for probabilities 0, 0·05, 0·1, 0·25, 0·5, 0·75, 0·90 and 1. Percentiles are depicted in figure 11 on straight lines parallel to the Y-axis by color marks. It can be seen in Figure 11 that the large starting variability of RPU is still increasing at time 4D to fall to minimum at 365D. B) Average perfusion units at each of the 11 time points. All 18 patients had at least one pre-operative scan indicating pathologic post-traumatic inflammation with an increase in perfusion (89); following grafting, inflammatory reduction is reflected as a relative decrease in perfusion that reaches a nadir between 6–14 days (PU 71–111 respectively), before a secondary increase in perfusion after 14 days that indicates engraftment and angiogenesis and peaks between 30–90 days (105 PU). Following this secondary peak and tissue regeneration, there is gradual drop in average perfusion to 61 PU at 360 days, indicating restoration of vascular quiescence.

Fig. 12

Clinical evaluation photography. Serial photographs on day 6, 14 and 30 post grafting. Each vertical row represents a single patient. Patient 1 was burned by melted metal on the job, and he was grafted on the 6th day post injury, 3rd degree burns covered 1·75% of TBSA. On the day of grafting the entire area was necrotic with full thickness damage. On the 6th day (A2) pink granulation tissue well-fixed to the wound bed, and devoid of secretion suggesting a vital skin grafts in over 99% of the surgically treated area. The blue arrowhead points to the Histoacryl® glue with which the graft is affixed to the tissue. Complete healing was achieved by 8 days (A3), and a month later a normally pigmented, non-hypertrophic scar devoid of any contractures was noted. Heal was the only hyperkeratotic area discolored by iodine. Patient 2 burned herself with day post injury. She had 3rd degree burns of 2·5% of TBSA on the right thigh, popliteal region a garden fire, and received the DESG/APC on 14th and calf of right leg (B1). On 6th day a vital skin graft was noted in over 99% of the surgically treated area. The wound was completely healed by 8 days, and one moth post-surgery (B4) a well-healed scar devoid of contractures, ulcerations or infections is evident. Patient 3 had Patient C burned herself while cooking on the hypothenar area of the palm and dorsum of 5th and 4th finger. She had 3rd degree burns on 1% of TBSA, but involving highly sensitive area of the left hand and fingers. She underwent grafting on the 8th day. On the 6th post-surgical day, fully vital skin grafts were noted on more than 99% of the surgical area. Complete healing was achieved on the 10th post-operative day. At one month the patient had a fully functional hand, and contraction-less scar.