INTRODUCTION
One of the requirements for successful cell-based therapy is the delivery of stem cells to target tissues after manipulations such as the expansion stem cell cultures or commitment of stem cells to a specific lineage. However, due to safety considerations such as transmission of viral or prion-related disease, the use of animal-derived products such as serum, tissue extracts, and enzymes in these manipulations is undesirable. So, several studies have examined the use of human-derived substitutes: Attempts were made to use human autoserum as a replacement for fetal bovine serum (FBS)1, though its volume available is limited. Patient-derived fibrin glue (thrombin and fibrinogen) and platelet-rich plasma have also been used for cell culture or in clinical trials for enhancing tissue regeneration2. Other studies have shown that growth factors derived from platelets can be used to stimulate cell proliferation3-5, but platelets can not provide some other major growth factors6.
The growth factor b-FGF, which is an important endogenous stimulator of angiogenesis7 and cell proliferation8, is released from surrounding wounded tissues during an early phase of wound healing9,10. Cellular b-FGF is released by the lysis of epidermal cells11, fibroblasts11, and endothelial cells12 around the wound, and b-FGF bound up in the extracellular matrix is released by the action of various wound proteases13,14. KGF is expressed in the dermis during wound healing15, and stimulates wound reepithelialization16. HGF was independently discovered as a powerful mitogen for hepatocytes and as a stimulator of dissociation of epithelial cells17. HGF-producing cells are found among those of mesenchymal origin, and HGF stimulates cell proliferation, cell migration18, and the production of matrix metalloproteinase (MMP)19 in keratinocytes.
Various growth factors directly or indirectly control phenomena accompanying wound-healing inflammation, remodeling and regeneration17, and can be detected in cutaneous wound fluids20-22, or in wound fluids obtained through surgical suction drains23-25. The fluid composition and the concentration of growth factors in the wound fluids change as healing progresses, and thus the fluids reflects the sequential wound healing phenomena. However, there is currently only limited information pertaining to the characterization of surgical drainage fluid with regard to growth factors and other soluble factors. Although a few previous reports have suggested that wound fluids have mitogenic and chemotactic effects8,26,27, there has been limited information beyond that. Also, although it may be clinically feasible to use wound fluids for cell-based regenerative therapies, protocols for use of wound fluids have yet to be optimized.
The present study focuses on the characterization of subcutaneous wound fluids obtained through surgical suction drains. Such fluids can be aseptically harvested with minimal morbidity: for example, adipose-derived stem cells can be isolated from liposuction aspirates and subcutaneous wound fluids can be simultaneously obtained by leaving a suction drainage tube in the subcutaneous cavity in the same surgery. We sequentially collected surgical drainage fluids from the subcutaneous space after plastic surgery, and characterized the fluids by examining wound healing-associated soluble factors such as electrolytes, cytokines, chemokines and MMPs. We compared these characterization profiles with those of platelet-poor and platelet-rich plasma, which can be also easily obtained from patients, and all three types of fluids were assessed for their potential utility in cell culture.
MATERIALS AND METHODS

Collection and preparation of human sera from platelet-rich plasma and platelet-poor plasma.
Human platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from four healthy volunteers. Blood was drawn into two 200 ml blood bags containing 0.327% citric acid, 2.63% sodium citrate, 0.0275% adenine, 0.251% sodium dihydrogenphosphate and 2.9% D-glucose solution (blood bag CPD-adenineR, Terumo, Tokyo, Japan). To isolate PRP, bags were centrifuged at 200 g for 10 min in a large-capacity refrigerated centrifuge (KUBOTA 9810, KUBOTA Co., Tokyo, Japan), and to isolate PPP the bags were centrifuged at 5000 g for 5 min; in both instances the supernatant was harvested. To obtain serum from PRP (designated SPRP) and from PPP (designated SPPP), 100 ml of PRP or PPP was drawn into a flask and 200 U of thrombin was added. The flask was agitated for 60 min at 37°C and then incubated overnight at 4 °C, after which the liquid component was drawn into 50 ml tube, centrifuged at 2000 g for 10 min, and supernatants were obtained as for SPRP and SPPP. The serum samples were frozen at -80°C and thawed at 37°C before analysis.

Collection and preparation of suction drainage fluid samples
We collected drainage fluids and punctured fluids from subcutaneous wounds from 15 patients, who underwent liposuction and abdominoplasty (7 patients), liposuction (5 patients), and breast augmentation (3 patients) at the University of Tokyo Hospital. Before samples were obtained, all patients gave signed informed consent, as approved by the ethical committee of University of Tokyo School of Medicine. Two to three weeks before surgery, 400 ml of blood was harvested from 10 of the 15 patients. The blood was separated by centrifugation into concentrated red blood cells and PPP; the cells were stored in preparation for autotransfusion, and the PPP was processed to obtain SPPP, using the previously described method. Suction drains (J-vac drainage system, Johnson & Johnson, Cornelia, GA, USA) were subcutaneously inserted into operative wounds during surgery, after which wound fluid was continuously suctioned at 40-60 mmHg and aseptically collected in the storage bag of the suction system. The wound fluids were centrifuged at 2,000 g for 30 minutes, and supernatant fluids were frozen at -80°C. The frozen samples were thawed at 37°C before analysis. Drainage fluid samples (N=52) obtained from 12 patients whose wounds exhibited normal healing (NH group), and punctured fluids (n=4) obtained from 3 patients who had subcutaneous seroma formation (SF group) were used in this study. Nineteen samples harvested on day 0 or day 1 from 12 patients of NH group were referred to as Drainage Fluid-Early (DF-E), while 9 samples harvested on day 5 or day 6 from 7 of the 12 patients of NH group were referred to as Drainage Fluid-Late (DF-L). Punctured fluid samples were harvested by puncturing the subcutaneous seroma on day 14 or later, and were included in our analyses as Punctured fluids (PF).

Biochemical analysis of serum and drainage fluid constituents
From each of 3 patients, we obtained a set of a preoperative serum samples and 7 sequential drainage fluids (day 0 to day 6). These samples (a total of 8 from each person) underwent biochemical analyses for total protein, albumin, sodium, potassium, chloride, calcium, and iron. Analysis was performed by SRL, Inc. (Tachikawa, Japan), a commercial analysis service.

Quantitative assays for cytokines, chemokines, and matrix metalloproteinases associated with wound healing
Concentrations of various cytokine growth factors (PDGF-BB, EGF, TGF-β1, b-FGF, VEGF, HGF, KGF and IGF-1) and chemokines (IL-6 and IL-8) in drainage fluid samples, punctured fluids, PRP, and PPP were assayed using anti-human ELISA kits (QuantikineR, R&D Systems Inc., Minneapolis, MN), according to the manufacturer’s instructions. Levels of immunoreactive cytokines as reported by the ELISA assay were measured at 450 nm by a microplate reader (Model 550, Biorad Laboratories, Hercules, CA), and a standard curve was generated to determine growth factor concentrations (pg/mL). Levels of MMP-1, MMP-8 and MMP-13 in drainage fluids were also measured using anti-human ELISA kits (Biotrak ELISA System, Amersham Biosciences, Piscataway, NJ).
Primary cell culture
Adipose-derived stromal cells (ASCs) were isolated from human lipoaspirates and cultured as previously described28. Briefly, the suctioned fat was digested with 0.075% collagenase in PBS for 30 min with agitation at 37oC. Mature adipocytes and connective tissues were separated from pellets by centrifugation (800 g, 10 min). Cell pellets were resuspended in erythrocyte lysis buffer (155mM NH4Cl, 10mM KHCO3, 0.1mM EDTA), incubated for 5 min at room temperature, resuspended again and passed through a 100-μm mesh filter (Millipore, MA, USA), and then plated at a density of 5 x 106 nucleated cells/100 mm plastic dish. Cells were cultured in M-199 medium containing 10% FBS at 37oC under 5% CO2 in a humidified incubator.
Human dermal fibroblasts were isolated from normal skin samples obtained from plastic surgery. The skin samples were cut into pieces of approximately 3 × 3 mm and treated with 0.25 % trypsin in PBS for 24 hour at 4°C. After removal of the epidermis, the connective tissue fragments were attached to 100 mm plastic dishes and cultured with DMEM containing 10% FBS. Primary fibroblasts appeared 4 to 7 days after the initiation of outgrowth cultures and became confluent after 2 to 3 weeks.
Cell proliferation assay using culture medium containing drainage fluids
Culture media (M199 for ASCs and DMEM for dermal fibroblasts) containing FBS and/or drainage fluids (DF-E [early] or DF-L [late]) was prepared at various concentrations. The drainage fluid samples were sterilized using a 0.22 μm filter (MILLEX GV Filter Unit, Millipore) before use. 5x104 cells were plated in 60mm dishes containing the prepared medium, and the medium was changed on the third and fifth days. The cell numbers were counted on the seventh day using a NucleoCounter (Chemometec, Allerod, Denmark), and average numbers were calculated from three different cultures of the cell types for each condition. The averages were normalized by calculating a ratio of cell numbers grown in each condition to cell numbers grown in the standard culture conditions (5% FBS without drainage fluid).
Statistics
The Kruskal- Wallis H- test with post hoc comparisons was used to compare concentrations of cytokines, chemokines and MMP among drainages fluids (DF-E and DF-L), punctured fluids, and SPPP. The Wilcoxon matched matched-pair t-test was used for comparison between SPRP and SPPP obtained from the same 4 volunteers, and also to compare cell numbers in each culture condition with that of control. P < 0.05 was considered to be significant.
RESULTS

Comparison of cytokine concentrations in SPRP and SPPP
Concentrations of various cytokine growth factors in SPRP and SPPP harvested from four volunteers are shown in Fig. 1. Concentrations of EGF, PDGF, TGF-β, VEGF and HGF were significantly higher in SPRP than in SPPP. The difference between the two serums was markedly seen in concentrations of EGF and PDGF. IGF-1 was similarly contained in both SPRP and SPPP, while bFGF and KGF were only minimally detected in both. PDGF detected in SPPP suggested a small amount of platelet contamination in SPPP.

Biochemical profiles of drainage fluids and blood serum
Concentrations of total protein and albumin in drainage fluids on day 0 were about 50% of those in preoperative serum, and both total protein and albumin gradually decreased to about 30% of that found in serum by day 6 (Fig. 2). Concentrations of Na+, K+, Cl-, Ca++, and Fe++ in drainage fluids were similar to those in serum and did not significantly change with time. The concentration of Ca++ in drainage fluids was about 60-70% of that in serum and changed very little from day 0 to day 6. The concentration of Fe++ in drainage fluid was extremely variable among different patients due to variations in an individual’s hemorrhage volume, but in general was substantially greater than that in serum, and tended to decrease slightly with time.

Cytokine concentrations in drainage fluids and SPPP
Daily sequential changes in various cytokine growth factors in drainage fluids from six (VEGF, KGF) or seven (all other cytokines) patients are shown in Fig. 3, and data of DF-E (n=19), DF-L (n=9), PF (n=4), and SPPP (n=10) from patients are summarized in Fig. 4. Concentrations of b-FGF, EGF, PDGF and TGF-β were much higher in DF-E than in DF-L or SPPP. SPPP contained much lower concentrations of VEGF, HGF, and KGF compared to DF-E, DF-L, and PF. PF contained high levels of TGF-β, VEGF, HGF, and KGF, while concentrations of b-FGF, EGF and PDGF were sufficiently low in PF that they could not be detected.
KGF concentrations peaked around day 3 and then began to decrease, in contrast to VEGF and HGF concentrations, which steadily increased with successive postoperative days. IGF-1 did not change significantly with postoperative time, and the concentration of IGF-1 in DF-E and DF-L was significantly lower (by approximately 50%) compared to SPPP.

Chemokine and MMP concentrations in drainage fluids, SPRP and SPPP
Daily sequential changes in the IL-6 and IL-8 chemokines and in MMPs (collagenases) were tracked in drainage fluids from seven patients, and the summarized data are shown in Fig. 5 and 6. IL-6 was present at high concentrations in DF-E and decreased gradually in successive postoperative days, while IL-8 increased gradually. DF-E contained twice as much IL-6 as was found in DF-L, while DF-L contained more IL-8 than DF-E. Neither IL-6 nor IL-8 was detected in SPPP. PF contained both chemokines, although there were not significant differences in PF chemokine levels vs. chemokine levels in either DF-E or DF-L (Fig. 5).
In drainage fluids, MMP-8 was present in much higher amounts as compared to MMP-1 and MMP-13 (Fig. 5). MMP-8 increased in the early phase of wound healing, peaked on day 2 ? 3, and then gradually decreased with time.

Cell proliferation assays using culture medium including the drainage fluids
In medium that had not been supplemented with drainage fluids, ASCs proliferated in a dose-dependent manner with regard to the concentration of FBS; the dose-dependent relationship was valid up to 10% FBS (Fig. 7A). When ASCs were grown in media containing 5% FBS, cell proliferation was significantly enhanced by the addition of DF-E or DF-L at concentrations of ?1%. The increase in proliferation was dose-dependent with respect to the concentration of DF-E or DF-L. When 5% FBS and 5% DF-E were added to the medium, cell count increased to over five times that of the control (5% FBS alone), and was twice as high as the cell count for media containing 10% FBS alone. In medium lacking FBS, the addition of drainage fluids significantly enhanced ASC proliferation but were less effective compared to the same concentrations of FBS. We also examined dermal fibroblasts, which proliferated in a dose-dependent manner with respect to FBS up to concentrations of 10% FBS (Fig. 7B). Although the proliferation of dermal fibroblasts was enhanced by drainage fluids in the absence or presence (5%) of FBS, enhancement of proliferation by the addition of drainage fluids was moderate. Judged from theBased on a comparison between 5% FBS+ 5% DF-E (or DF-L) and 10% FBS, drainage fluids showed no additionalexhibited no value as an additive to FBS on in terms of expansion of dermal fibroblasts.

DISCUSSION
Biochemical analysis of drainage fluids
Results from the analysis of the biochemical composition of drainage fluids in this study were similar to those of a previous study analyzing biochemical profiles of drainage fluids after axillary dissection29. Our analysis showed that concentrations of Na+, K+, and Cl- in drainage fluids were similar to those in plasma, while concentrations of Ca++, total protein and albumin in drainage fluids were ~60 ? 80% lower than those in plasma. The concentration of Fe++ in drainage fluid was generally higher than in plasma but was variable, (depending on the hemorrhage volume) and decreased over time.
Extracellular fluid volume, which makes up approximately 20% of body weight, is composed of 5% plasma (or intravascular fluid) and 15% interstitial fluid. The concentrations of Na+, K+, and Cl- in interstitial fluid are similar to those in plasma, while the total protein concentration of interstitial fluid is less than a third of that of plasma30. It is therefore likely that our drainage fluids consisted primarily of interstitial fluid, with plasma composing the remaining ~20-40% of the total volume. However, drainage fluids are not simply a mixture of plasma and interstitial fluids because unlike these fluids, they also contain several other types of proteins, including various cytokine growth factors and chemokines.

Cytokine growth factor profiles in drainage fluids
The sequential changes in cytokine profiles in drainage fluids shown in this study clearly reflected the successive activities of various cells and the sequential phenomena involved in the wound healing process (Fig. S1). In the early phase (postoperative days 0-1) of wound healing, b-FGF, PDGF, EGF, and TGF-β1 were present at high concentrations, and levels subsequently decreased acutely in the next stage of wound healing. Since b-FGF is known to be primarily derived from injured tissue or from cells infiltrating into wounds at early stages, tissue-bound b-FGF may be released after injury by several mechanisms, including cell lysis and cell injury11,12,31. PDGF, EGF, and TGF-β1 were detected at higher amounts in SPRP than in DF-E, suggesting that these growth factors were mainly supplied by dying, lytic platelets in the early phase of wound healing.
In the second phase of wound healing (postoperative days 2-4), KFG concentrations peaked, and those VEGF and HGF slightly increased. Later, in the third phase, (days 5-6) VEGF and HGF concentrations gradually increased to peak levels. Since these growth factors were present at only very low levels on days 0-1, it is possible that these increases resulted from their release from the cells that migrated to the wound site after day 1. KFG, VEGF, and HGF are thought to have roles primarily in granulation, angiogenesis and epithelialization32-34. KGF is known to be released mainly from fibroblasts and T cells35,36, VEGF from keratinocytes and macrophages37,38, and HGF from mesenchymal cells such as dermal fibroblasts18.
Punctured fluids from subcutaneous seroma contained higher concentrations of growth factors seen in later phases such as VEGF, HGF, and KGF, but TGF-β1, which is seen in the early phase, was also abundant in seroma fluid. This finding may be based on different sources of the early-phase growth factors: PDGF and EGF are mostly derived from platelet, whereas TGF-β1 is supplied not only from platelets but also from various sources. The initial production of active TGf-β1 from platelets serves as a chemoattractant for neutrophils, macrophages, and fibroblasts, and these cells further enhance TGF-β1 production17.

Chemokine and MMP profiles in drainage fluids
In our study, IL-6, a major mediator of the host response to tissue injury39, was present in DF-E at about 7000 pg/mL and gradually decreased afterwards. Cells that appeared in the wound area in each phase seemed to be major sources of IL-6; neutrophils were present in the early phase and macrophages and lymphocytes were present in later phases. In the case of IL-8, the concentration gradually increased up to day 6, so it is probable that the fibroblasts present in the second wave of cell migration to the wound produced significant amounts of IL-8, as was previously suggested in a study of fetal wound healing40.
Pro-inflammatory cytokines/chemokines directly stimulate the synthesis of the collagen-degrading matrix metalloproteinases (MMPs) and also inhibit the synthesis of tissue inhibitors of metalloproteinase in fibroblasts and endothelial cells41. Fibroblasts appear to be the cellular source for the majority of MMP-142, while neutrophils seem to provide most of MMP-843. In subcutaneous drainage fluids, MMP-1 gradually increased up to day 6, while MMP-8 peaked on days 2 to 3. At all time points examined, levels of MMP-8 were statistically significantly higher than both MMP-1 (50-fold to 200-fold) and MMP-13 (1000-fold to 10000 fold). The sequential changes that we observed in MMP-1 and MMP-8 were similar to previously reported data from a study of cutaneous wound fluids44. Taken together, these data suggest that MMP-8 functions as the primary debriding collagenase during the acute phase of wound healing.

Potential use of wound fluids and SPRP in culture media
Since our data showed that drainage fluids contained various growth factors which were not found in SPPP or SPRP, we tested drainage fluids as a substitute or supplement for serum in the culturing of ASCs and dermal fibroblasts. The experiment using ASCs showed that DF-E is superior to FBS as a 5% additive of the medium containing 5% FBS, while that using dermal fibroblasts suggested that drainage fluids may be used as similarly to serum. Thus, we suggest that drainage fluids may be used as a supplement or substitute for serum in culture media, and may be able to support the growth of cell types other than the two lines examined in this study.
Recent developments in the clinical use of cultured cells (such as stem cell therapy or gene delivery) have necessitated safer preparation and manipulation of cells, which partly entails avoiding the use of animal-derived serum, tissues and extracts. In this respect, autologous serum, cytokines or other soluble factors could be extremely valuable. For example, ASCs isolated from liposuction aspirates of a patient could be cultured using the patient’s own SPRP taken from blood and/or using drainage fluids taken from the subcutaneous wound after liposuction. Subcutaneous wound fluids have some advantages compared to cutaneous and intraperitoneal wound fluids: cutaneous wound fluids are difficult to collect aseptically and in a large volume, and intraperitoneal ones can be obtained only through major abdominal surgery and are not aseptic in most cases.
The present results could be used as a guide in choosing the appropriate fluid supplement for cell culture, based on the specific needs of a given cell line (Table. S1). Both SPRP and drainage fluids are economical ready-made mixtures of serum (plasma) and soluble factors such as cytokines, and can also be used as raw materials for the extraction of individual soluble factor proteins. Further investigation will be necessary to provide optimized protocols for the usage of drainage fluids and SPRP in cell culture and in factor isolation.

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Figure Legends

Fig. 1
Comparison of cytokine concentrations in SPRP and SPPP.
Serums from platelet-rich plasma (SPRP) and from platelet-poor plasma (SPPP) were collected from four volunteers. Cytokine concentrations were measured in SPRP and SPPP. Values represent means ± S.E. * P<0.05 (blue lines), ** P<0.01 (red lines).

Fig. 2
Biochemical profiles of preoperative serum and of drainage fluids on days 0 to 6.
Preoperative serum and drainage fluids were collected from the same three patients; drainage fluids were collected on days 0-6, where day 0 represents the day that surgery was performed. The preoperative serum value is indicated to the left on the x-axis and is labeled “serum”; the numbers 0-6 to the right represent day 0 through day 6 timepoints of drainage fluid collection. Values represent means ± S.E.

Fig. 3
Daily changes in cytokine concentrations in drainage fluids.
Drainage fluids were collected on days 0 to 6 from patients in the normal healing (NH) group, and cytokine concentrations were examined by ELISA. In some patients, drainage fluids were not obtained on all the designated days. For VEGF and KGF, data was derived from six patients; for all other cytokines, data was derived from seven patients. Each line shows data from one patient (e.g. 38F means 38 year-old female).

Fig. 4
Comparison of cytokine concentrations in DF-E, DF-L, PF, and SPPP.
Cytokine concentrations were measured in drainage fluid samples from day 0 or day 1 (DF-E; Drainage Fluid-Early), drainage fluid from day 5 or day 6 (DF-L; Drainage Fluid-Late), punctured seroma fluid samples from days 14+ (PF; Punctured Fluids), and serum from platelet-poor plasma (SPPP). Values represent means ± S.E. * P<0.05 (blue lines), ** P<0.01 (red lines).

Fig. 5
Changes in IL-6 and IL-8 concentrations in DF-E, DF-L, PF, and SPPP.
The concentration of IL-6 and IL-8 was measured by ELISA in drainage fluids collected on days 0-6 and was similarly measured in PF and SPPP. Data of daily changes from seven patients were shown in left figures, in which each line shows data from one patient (e.g. 38F means 38 year-old female). Values in right graphs represent means ± S.E. * P<0.05 (blue lines), ** P<0.01 (red lines).

Fig. 6
Changes in concentrations of MMP-1, MMP-8, and MMP-13 in DF-E, DF-L, PF and SPPP.
The concentration of MMP-1, MMP-8, and MMP-13 was measured by ELISA in drainage fluid collected on days 0-6 and in seroma puncture fluid (PF), SPPP, and SPRP. Data of daily changes from seven patients were shown in left figures, in which each line shows data from one patient (e.g. 38F means 38 year-old female). Values in right graphs represent means ± S.E. * P<0.05 (blue lines).

Fig. 7
Proliferation of ASCs and dermal fibroblasts.
ASCs (A) and dermal fibroblasts (B) were cultured with DMEM containing various amounts of FBS and/or drainage fluids (DF-E or DF-L) for 1 week and cell numbers were counted. Each cell number was expressed as a ratio to that of the control culture, which was grown in media that contained 5% FBS and lacked drainage fluid. Values represent means ± S.E. * P<0.05 (bracketed blue lines), ** P<0.01 (bracketed red lines).

Fig. S1

Summary of sequential changes in soluble factors associated with wound healing in drainage fluids from subcutaneous wounds.
There are three types of sequential changes in the abundance of soluble factors that function in wound healing. First, levels of b-FGF, EGF, PDGF and TGF-β are initially high and then gradually decrease. EGF and PDGF in drainage fluids in the early phase (coagulation phase) of wound healing are derived from platelets, although TGF-β is derived from various sources, and b-FGF is mainly derived from injured tissue or from cells infiltrating into wounds at early stages. Second, KGF, IL-6, and MMP-8 peak around days 2 to 4 (during the inflammatory phase). KGF is released from T lymphocytes and fibroblasts, while IL-6 seems to be discharged from the various cells involved in each phase. Third, VEGF, HGF, IL-8, and MMP-1 are low in the early phase and gradually increase up to the late phase (proliferation phase). These factors are derived from cells involved in the later phases of wound healing, including fibroblasts and keratinocytes. IGF-1 is present at relatively consistent levels throughout the entire wound healing process. MMP-13 is detected only in minimal quantities.

Supplement table

　 drainage fluids serum best source
　 DF-E DF-L SPPP SPRP
　 　 　 　 　 　
bFGF (++) (-) (-) (-) DF-E
EGF (+) (-) (-) (++) SPRP
PDGF (+) (-) (-) (++) SPRP
TGF-β (+) (-) (-) (++) SPRP
VEGF (+) (++) (-) (-) DF-L
HGF (+) (++) (-) (-) DF-L
KGF (+) (++) (-) (-) DF-L
IGF-1 (+) (+) (++) (++) SPPP, SPRP
IL-6 (++) (+) (-) (-) DF-E
IL-8 (+) (++) (-) (-) DF-L
MMP-1 (±) (+) (±) (+) DF-L, SPRP
MMP-8 (++) (+) (-) (-) DF-E
MMP-13 (-) (-) (-) (-) -
　 　 　 　 　 　

Table S1
Sources of autologous soluble factors associated with wound healing: comparison of drainage fluids, SPPP and SPRP.
Relative abundances are indicated by -, +/-, +, and ++. ++ indicates high abundance of a factor, and ? indicates absence of a factor.