Umbilical cords and amnion from four full-term (38–40 weeks) healthy pregnancies (excluding HIV, hepatitis and syphilis) through cesarean section were obtained from the Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Shantou University Medical College (Shantou, China). Informed consent and ethical permission were obtained before parturition in accordance with the Ethics Committee of Shantou University Medical College.

Isolation, culture and identification of HUMSCs was performed in the translational medicine center, as previously described (15). Briefly, within 24 h after collection of umbilical cords from the operating room, umbilical cords were flushed with phosphate buffered solution (PBS) to remove blood cells. Umbilical cords containing two arteries and a vein buried within the mucous connective tissue, known as the Wharton's jelly, were sectioned into pieces (3–4 cm long) with a sterile scalpel. After removing intact vessels, the remaining Wharton's jelly was transferred to a sterile container containing high glucose Dulbecco's modified Eagle's medium (H-DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then dissected into smaller pieces. Thereafter, explants were plated on 100 mm cell culture dishes (JET BIOFIL) which were inverted in a humidified atmosphere with 5% CO₂ at 37°C for 15–30 min until the explants adhered to the bottom of the dish. Fresh growth medium [5 ml; H-DMEM supplemented with 10% fetal bovine serum, 100 mg/ml penicillin, 100 mg/ml streptomycin and 1 mg/ml amphotericin B (all Gibco; Thermo Fisher Scientific, Inc.)] was then added to cover the explants. Cell culture dishes were left undisturbed at 37°C in a humidified incubator at 5% CO₂. The culture medium was replenished every 2 days until sporadic fibroblast-like cells grew from the tissue edge, usually after 5–7 days. Cells were passaged when cultures reached 80–90% confluence. Third passage cells were used in experiments. HUMSCs at passage 3 were characterized using flow cytometry to examine the expression of pluripotent cell markers as previously detailed (15).

CM-DiI is a lipophilic carbocyanine fluorescent dye, which attaches to the cell membrane and has low cytotoxicity. CM-DiI can be well preserved after fixation, permeabilization and paraffin embedding of cells. Therefore, CM-Dil is been widely used for immunofluorescence staining on tissue sections (17). HUMSCs at passage 3 were cultured in complete growth medium under aforementioned conditions. Prior to labeling, all HUMSCs were suspended at a density of 1×10⁶/ml in serum-free DMEM and incubated with 4 µMCM-DiI (Molecular Probes; Thermo Fisher Scientific, Inc.) for 15 min at 37°C, followed by 10 min incubation at 4°C to optimize staining levels. Cells were then precipitated at 250 × g for 5 min at 37°C. Cell pellets were gently re-suspended in 37°C medium and washed twice. The efficiency of CM-DiI labeling was checked by immunofluorescence using fluorescence microscopy (Olympus BX51, Olympus Corporation, Tokyo, Japan). Briefly, the cells seeded onto coverslips and fixed in 4% paraformaldehyde for 30 min and then washed with PBS three times. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min as a nuclear stain. Positive cells were scored from four randomly selected fields per specimen, and the efficiency of CM-DiI labeling was represented as incorporation into the positive rate. Viability of CM-DiI labeled HUMSCs was assessed by Trypan blue staining.

The preparation of acellular amnion was performed as detailed previously (18). Briefly, human amnion collected at cesarean section was washed with PBS under sterile conditions and stored at −80°C in PBS containing 12% dimethyl sulfoxide. The amnion was frozen and thawed 3 times and cut into pieces (2.5×2.5 cm), followed by the removal of epithelial cells by incubation in 0.02% EDTA at 37°C for 2 h using gentle scraping under a microscope. The spongy layer was also removed. The complete removal of the epithelial cells was confirmed by hematoxylin and eosin (H&E) staining.

PPP was the residual material of platelet rich plasma (PRP) therapy collected following cosmetic surgery procedures on outpatients of the Second Affiliated Hospital, Shantou University Medical College. Informed consent was obtained from the patients who provided PPP before the collection procedure. The specific extraction method was performed as previously described (19). Blood samples were centrifuged twice to obtain PPP and PRP. A 30 ml venous blood sample was collected under aseptic conditions at room temperature, and was aspirated with a 21 G needle into a 50 ml sterile centrifuge tube preloaded with 6.5 ml anticoagulant citrate dextrose solution. The blood sample was centrifuged for 15 min at 320 × g, at 4°C, resulting in the following three layers: An inferior layer composed of red cells, an intermediate layer composed of white cells, and a superior layer made up of plasma. The 20 ml plasma layer was centrifuged for a further 5 min at 1,000 × g in order to obtain a two-part plasma sample: The upper part, consisting of 18 ml PPP; and the lower part, consisting of 2 ml PRP. The PPP was gently aspirated with a pipette and placed in a sterile centrifuge tube, without being mixed with PRP. Allergic reactions were not observed when administering PPP in these experiments.

When passage 3 CM-DiI labeled HUMSCs reached 80% confluence, HUMSCs were detached with trypsin solution, washed with PBS (37°C) and precipitated. Cell pellets were re-suspended in PPP at a density of 1×10⁶/ml, and 1 ml PPP was transferred to each well of a 12-well tissue culture plate (Corning Incorporated, Corning, NY, USA), followed by addition of 10% calcium gluconate at a volume ratio of 1:7. Preliminary experiments revealed that this ratio was the most conducive to forming PPP gels (data not shown). Plates were left undisturbed for 15 min at 37°C in a humidified incubator with 5% CO₂ to allow PPP gel formation. Following gel formation, fresh growth medium was added to cover the gel to maintain HUMSC growth. Gels containing HUMSCs were used in wound healing in animals the following day. Blank gels were made in the same way, without the inclusion of HUMSCs, and were used as a control. At the time of surgery, all gels were detached from the 12-well plates with forceps (Fig. 1A).

As HUMSCs possess immunosuppressive properties, Sprague-Dawley (SD) rats were used rather than severe immune deficient mice. SD rats (age, 16 weeks; weight, 240–260g; n=36) were purchased from Shantou University Medical College Laboratory Animal Center (SUMC; Shantou, China) and maintained under specific pathogen free conditions in the Laboratory Animal Center of SUMC. All animals were allowed to acclimate for 1 week in the facility before experiments were performed. Animal protocols were approved by the Institutional Animal Care and Use Committee at SUMC.

SD rats were randomly assigned into four groups (n=9 rats/group): Group 1, grafted with PPPA+HUMSCs (PPPA+cells; PPPAC); group 2, grafted with PPPA (PPPA); group 3, injected with HUMSCs (Injection); and group 4, injected with PBS as blank control (Blank). An excisional wound splinting mouse model was generated as previously described (20) with slight modifications. In brief, after hair removal from the dorsal surface under anesthesia (10% chloral hydrate solution; 0.1 ml/20g; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), two 14 mm (area 1.5386 cm²) full thickness excisional skin wounds were created on each side of the midline. A donut-shaped silicone splint was placed so that the wound was centered within the splint, which was fixed to the skin by an immediate-bonding adhesive. Interrupted sutures were utilized to stabilize its position. The same shaped hoops were attached to the silicone splint to prevent rats from gnawing it. Acellular amnion was placed over the wounds. After the surgical procedure, the animals were housed individually. Our preliminary studies suggested that the adhesive on the skin in rats prior to the experiment did not cause any skin irritation or allergic reaction. Each wound in the PPPAC group was grafted with PPP gel with 1×0⁶ CM-DiI labeled HUMSCs (Fig. 1B-D). In the PPPA group, the same procedure was employed for the PPPAC group, except for the use of the blank gels. Each wound in the Injection Group received 1,000 µl HUMSC suspension containing 1×10⁶ CM-DiI-labeled HUMSCs, among which 800 µl was used for subcutaneous injection around the wound and 200 µl for topical application on the wound bed. Blank Group animals were injected with PBS as a blank control.

Following surgery, wound dressings were changed every day and the behavioral activity and wound healing progression of the SD rats was monitored. On days 0, 2, 4, 6, 8, 10, 12 and 14 post-surgery, the open wounds on both sides of midline of each animal from the four groups were imaged and wound healing was assessed by comparison of images captured at different time points with the initial images using an image analyzer (Image Pro Plus 6, Media Cybernetics, Inc., Rockville, MD, USA). The healing index included re-epithelialization and contraction. The healing rate was calculated as (area of original wound-area of left exposing wound)/area of original wound ×100%.

In addition, 2 SD rats randomly selected from each group were sacrificed at 3, 7 or 14 days, and the skin samples containing the wound and 3 mm of the surrounding skin were harvested (21), fixed in 10% buffered formalin and embedded in paraffin. H&E staining was performed on 3-µm thick tissue sections.

Sequential slides were used for detecting the distribution of CM-DiI- labeled HUMSCs by immunofluorescence. Briefly, the sections were rehydrated and the antigens were retrieved via microwaving in 10 mM sodium citrate (pH 6.0) for 10 min, followed by counterstaining with DAPI. Following this, slides were washed 3 times with PBS, covered with anti-fade solution, and positive cells were scored on the wound region under fluorescence microscopy to evaluate the distribution of HUMSCs.

The same procedure described above was used to generate PPPA. In the experimental group (PPPAC group), passage 3 PPPA HUMSCs (a total of 1×10⁶ CM-DiI labeled HUMSCs in 1 ml PPP) grown in a 12-well plate, were cultured in vitro, and the supernatant of each well in experimental group was collected on days 1, 3, 7 and 14. In the control group (PPPA group), PPPA without HUMSCs formed in a 12-well plate were cultured in vitro, and the supernatant was collected on day 1 as an initial reference. A monolayer group (a total of 1×10⁶ CM-DiI labeled HUMSCs in 2D culture conditions in 1 well of a 12-well plate) were used as a 2D reference (2D group). The culture medium was changed 24 h prior to supernatant collection, and the expression levels of growth factors including insulin-like growth factor (IGF-1), hepatocyte growth factor (HGF), transformation growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF) in the collected supernatant was detected using ELISA kits (Human VEGF Quantikine ELISA kit; cat. no. SVE00; Human HGF Quantikine ELISA kit; cat. no. SHG00; Human KGF Quantikine ELISA kit; cat. no. DKG00; Human TGF-β1 Quantikine ELISA kit; cat. no. SB100B; and Human IGF-1 Quantikine ELISA kit; cat. no. SG100) from R&D Systems, Inc. (Minneapolis, MN, USA) according to the manufacturer's protocol. The data were compiled from three independent assays per sample with each performed in duplicate.

Data were analyzed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA) and are presented as the mean ± standard deviation. Student's paired t-test was performed for comparison of data of paired samples. One-way analysis of variance was used for multiple group comparisons followed by the least significant differences t-test. P<0.05 was considered to indicate a statistically significant difference.

Fig. 2 presents the primary culture of isolated HUMSCs. At day 5–7 in culture, spindle-shape cells (Fig. 2A, black arrow) began to migrate out from Wharton's jelly fragments (Fig. 2A, white arrow). Half of the volume of culture medium was replaced with new fresh medium every 3–4 days until cells completely covered the culture area, which was reached by ~day 10–14. HUMSCs exhibited fibroblast-like morphology, with most demonstrating a flat, wide and polygonal appearance after passaging. The above-mentioned morphology did not alter significantly up to passage 9 (Fig. 2B-D). Cells at passage 3 expressed cluster of differentiation (CD)29, CD59 and CD44, but not CD34 and human leukocyte antigen D related (data not shown), and were used in the subsequent experiments.

CM-DiI labeled 98.4% HUMSCs isolated from human umbilical cord as determined by immunofluorescence (Fig. 3), while only <5% CM-DiI-positive HUMSCs were trypan blue positive (17) (data not shown). Further culture revealed that CM-DiI labeling, present in the cytoplasm and not the nucleus, did not significantly affect the proliferation of HUMSCs (data not shown). CM-DiI-labeled fluorescence was clearly visible at 14 day after culture.

In the animal wound model used in this study, it was demonstrated that PPPA significantly enhanced HUMSC-mediated wound healing in SD rats (Table I). From day 4 post-surgery, the healing rate of the PPPAC group was significantly higher compared with the other three experimental groups (Table I; P<0.05 and P<0.01). However, at days 2, 6, 8 and 14, the difference in the healing rate between the PPPA group and the Injection group was not statistically significant (Table I). Wound contraction was markedly reduced in the PPPAC and PPPA groups compared with the injection and blank groups (Fig. 4A). Furthermore, the thickness of the newly formed epidermis layer of the PPPAC group grew faster to cover the wounded skin tissue compared with the other groups either on the 7th or the 14th day, as examined by histology. In addition, the dermal papillae and the amount of CM-DiI-labeled HUMSCs in the regenerated skin tissue were significantly increased in the PPPAC group. The alignment of fibers in the healing skin tissue appeared more regular in the PPPAC group than the other groups (Fig. 4B). Therefore, it was concluded that the PPPAC group demonstrates superior wound healing compared with the other three groups.

Next, the present study determined the distribution of CM-DiI labeled HUMSCs in the wounded area of skin of rats from the different experimental groups. Immunofluorescence was observed in the basal layer of epidermis, dermis, spinous layer and superficial fascia in newly regenerated skin tissue in the PPPAC and Injection groups (Fig. 5 and data not shown). Fluorescent-positive cells were polygonal or spherical in shape and were located in the newly regenerated skin tissue or in the nearby healthy skin tissue. The distribution of HUMSCs in PPPAC group was more homogeneous compared with that in PPPA group. Taken together, these results indicated that the PPPAC group demonstrates a better survival rate of HUMSCs after engrafting.

ELISAs were subsequently performed to evaluate the protein expression levels of secreted growth factors (including HGF, TGF-β1, VEGF, IGF-1 and KGF) in HUMSCs at different culture stages and times in the PPPA group (Fig. 6). As PPPA contained certain baseline levels of growth factors, the culture medium was collected at day 1 from the PPPA group as an initial reference value. The findings suggested that the secretion levels of HGF, TGF-β1, VEGF, IGF-1 and KGF released by HUMSCs in the PPPAC group were significantly increased compared with those from the 2D group. Therefore, it was concluded that PPPAC promotes the release of growth factors from HUMSCs after engrafting into the wounded area of skin.