[Research advances on the role of microRNA engineered exosomes in diabetic wounds].

Diabetic wounds are a common complication in patients with diabetes, which is difficult to treat. Current treatment methods for diabetic wounds include debridement, functional dressing coverage, negative pressure therapy, bone cement filling, and skin grafting, etc. MicroRNA (miRNA) engineered exosomes have shown promising potential in diabetic wound repair due to the ability to alleviate inflammation, stimulate angiogenesis, and promote collagen deposition and re-epithelialization. Related researches are being actively carred out. This paper reviews the pathophysiological characteristics of diabetic wounds, the characteristics of miRNA and exosomes, the engineering methods for exosomes loaded with miRNA, and the mechanism of miRNA engineered exosomes in promoting healing of diabetic wounds, aiming to provide a reference basis for the future clinical application of miRNA engineered exosomes in diabetic wounds.

[Clinical effects of antibiotic bone cement combined with free anterolateral thigh flap in sequential treatment of diabetic foot ulcer].

Objective: To investigate the clinical effects of antibiotic bone cement combined with free anterolateral thigh flap in sequential treatment of diabetic foot ulcer (DFU) wounds. Methods: A retrospective observational study was conducted. From August 2018 to August 2021, 15 patients with DFU who met the inclusion criteria were admitted to the Affiliated Hospital of Zunyi Medical University, including 12 males and 3 females, aged 42-65 years, with a history of type 2 diabetes for 5-19 years. All the wounds of patients were complicated with local bone, muscle, or tendon defects or exposure. The wounds were covered with antibiotic bone cement after debridement in stage Ⅰ+free anterolateral thigh chimeric perforator flap (perforator flap+muscle flap) or simple free anterolateral thigh flap grafting in stage Ⅱ. The defect area of the wound after bone cement removal and debridement was 9.0 cm×5.0 cm-20.0 cm×7.0 cm, the incision area of the flap was 10.0 cm×5.0 cm-22.0 cm×7.0 cm, and the incision area of the muscle flap was 5.0 cm×3.0 cm-8.0 cm×4.0 cm. The donor sites of flaps were sutured directly. During follow-up, the situations of donor site healing and flap survival were observed. At the last follow-up, the texture and shape of the flap, the presence of new ulcers on both limbs, and the walking ability of the patient were observed. Results: During the follow-up of 8 to 21 months after operation in stage Ⅱ, the donor sites healed well with only residual linear scar; flaps in 14 patients survived completely, and the flap in 1 patient developed partial necrosis at 3 weeks after stage Ⅱ surgery, which was healed after debridement and skin grafting. At the last follow-up, the flaps were good in texture and appearance, there were no new ulcers in the affected limb or opposite limb, and the patients had no obvious impairment in daily walking function. Conclusions: To repair DFU wounds with antibiotic bone cement combined with free anterolateral thigh flap can rapidly control the infection, achieving a high survival rate of flap after operation with no obvious impairment in daily walking function of patients.

[Clinical application of combination of different types of free perforator flaps in the repair of complex wounds in extremities].

Objective: To investigate the clinical application effects of combination of different types of free perforator flaps in the repair of complex wounds in extremities. Methods: A retrospective observational study was conducted. From January 2018 to June 2022, 11 patients with complex wounds in extremities who met the inclusion criteria was admitted to the Affiliated Hospital of Zunyi Medical University, including 8 males and 3 females, aged 28 to 55 years. The wounds in the upper extremities in 4 cases and in the lower extremities in 7 cases were repaired with different combination of free perforator flaps. After debridement, the wound area was 7.0 cm×6.0 cm-28.0 cm×12.0 cm. A combination of different types of perforator flaps were applied, including the perforator tri-leaf flap of the descending branch of the lateral femoral circumflex artery in 6 cases, the descending branch of lateral femoral circumflex artery combined with oblique branch perforating branch flap in 2 cases, the lobulated flap of the descending branch of the lateral femoral circumflex artery combined with the contralateral medial plantar artery perforator flap in 2 cases, and the bilateral perforator flap of the descending branch of lateral femoral circumflex artery combined with great toe nail flap in 1 case, with the size of a single flap ranged from 2.0 cm×2.0 cm-25.0 cm×6.0 cm. The donor site was repaired by direct suture, skin grafting, or flap transplantation. During free flap transplantation, the flap was cut and split according to the distribution of perforators, and end-to-end or end-to-side anastomosis was performed between the donor area and the recipient area. After surgery, the survival of transplanted flap in the primary recipient site, the occurrence of vascular crisis, the wound healing in the flap donor site, and the survival of transplanted skin or flap in the flap donor site were observed. During follow-up, the blood supply, appearance and texture of the transplanted flap in the primary recipient site were observed; and at the same time, the weight bearing of the plantar receiving area, the presence of sliding, ulcers, and sinus tracts of the flap, and the appearance and function of the hand were observed; the complications in the donor area were observed. Results: After surgery, one patient's transplanted flap in the primary recipient site had vascular crisis but survived after exploration+vein graft bridging; partial necrosis occurred in one lobe of anterolateral thigh lobulated flap transplanted to the primary recipient site in one patient and recovered after dressing change+skin grafting, and the different types of perforator flap transplanted to the primary recipient site in the other 9 patients all survived. After surgery, the wound with direct suture at the donor site healed well, and the skin or flap transplanted to the donor area survived well. During 3-24 months of follow-up, the blood supply, appearance, and texture of the transplanted flap at the primary recipient site were good. In two patients, the anterolateral thigh flap combined with the medial plantar flap were used to repair plantar defects. The plantar receiving area was able to bear weight, and the texture of the flaps in the recipient area was close to the normal plantar skin, without flap sliding, ulcer, or sinus tract formation. In one patient, bilateral anterolateral thigh flap combined with great toe nail flap were used to repair hand combined with soft forearm defect, and the appearance and function of hand, especially thumb were good. Only linear scar was left in the donor site without other obvious complications. Conclusions: The combination of different types of perforator flaps is a reliable clinical method to repair complex wounds in extremities with high safety, good efficacy, and less complications.

[Free anterolateral femoral muscle flap and perforator flap transplantation for repair of the huge wound after periorbital tumor resection and orbital enucleation].

Objective: To observe the clinical effect of free anterolateral femoral muscle flap and perforator flap transplantation for repair of the huge wound and after periorbital tumor resection and orbital enucleation. Methods: It was a retrospective case series study. Twelve patients with orbital tumors admitted to the Department of Burn and Plastic Surgery of the Affiliated Hospital of Zunyi Medical University from February 2017 to April 2021 were included. There were 4 males and 8 females, aged 48 to 87 years. Nine patients had cutaneous squamous cell carcinoma, and 3 had basal cell carcinoma. All patients underwent extended resection of the tumor, resection of orbital contents and wound repair.All patients had the lesion completely removed, chimeric anterolateral thigh flap of the anterolateral femoral flap and perforator flap were transplanted to repair the wound. The donor area of the flaps was closed with tension sutures. The size of intraoperative resection lesion,intraoperative resection flap and muscle flap and the depth of the wound cavity were summarized. The postoperative flap survival, wound healing, surgical area appearance, flap color, thickness and texture, scarring and sensation in the surgical area, and tumor recurrence were observed. Results: The surgical procedures were successfully completed in all the 12 patients. The intraoperative resection lesion ranged from 7.0 cm × 5.0 cm to 15.0 cm × 8.0 cm. The depth of the wound cavity was 4.0 to 5.0 cm. The intraoperative resection flap range was 7.0 cm × 5.0 cm to 19.0 cm × 8.0 cm. The muscle flap size ranged from 4.0 cm × 3.0 cm to 5.0 cm × 4.0 cm. All flaps completely survived after surgery, and the wounds healed. The sutures at the recipient area were removed at 5 to 7 days after surgery, while the sutures at the donor area were removed at 12 to 14 days. All of the patients were followed up for 3 to 30 months. The scar at the periorbital area was concealed, and the color, thickness and texture of the flaps were similar to those of the surrounding normal skin. The scarring in the flap supply area was not hypertrophic, with localized decreased skin sensation around it. None of the patients had any tumor recurrence during the follow-up period. Conclusion: The anterolateral femoral muscle flap and perforator flap transplantation could efficiently repair the huge wound after orbital content removal, achieving satisfactory therapeutic effects.

[Research advances on mesenchymal stem cells and their derivatives in the treatment of acne scars].

Acne is a common chronic inflammatory disease of the skin that often occurs on the face, and acne scars are often secondary to the healing process of acne, which often leads to impaired appearance and psychological disorders of patients. The current treatment for acne scars is extremely difficult. With the development of regenerative medicine, stem cell transplantation has become a new treatment for acne scars. In recent years, it has been reported that stem cells and their derivatives can effectively antagonize the formation of acne scars. Therefore, this paper briefly reviews the basic and clinical researches on the treatment of acne scars with various mesenchymal stem cells and their derivatives, aiming to provide theoretical basis and reference for the stem cell therapy of acne scars.

[Clinical application effects of two longitudes three transverses method in perforator location of thoracodorsal artery perforator flap and deep wound repair].

Objective: To explore the clinical application value of two longitudes three transverses method in the location of the perforator of thoracodorsal artery perforator and deep wound repair. Methods: The retrospectively observational study was conducted. From December 2018 to June 2020, 17 patients with deep wounds who were admitted to the Affiliated Hospital of Zunyi Medical University met the inclusion criteria and were included in this study, including 7 males and 10 females, aged 12 to 72 years. The wound areas of patients after debridement were 7 cm×3 cm to 11 cm×7 cm. Two longitudinal lines were located through the midpoint of the armpit, the posterior superior iliac spine, and the protruding point of the sacroiliac joint, and three transverse lines were located 5, 10, and 15 cm below the midpoint of the armpit between the two longitudinal lines, i.e. two longitudes three transverses method, resulting in two trapezoidal areas. And then the thoracodorsal artery perforators in two trapezoidal areas were explored by the portable Doppler blood flow detector. On this account, a single or lobulated free thoracodorsal artery perforator flap or flap that carrying partial latissimus dorsi muscle, with an area of 7 cm×4 cm to 12 cm×8 cm was designed and harvested to repair the wound. The donor sites were all closed by suturing directly. The number and location of thoracodorsal artery perforators, and the distance from the position where the first perforator (the perforator closest to the axillary apex) exits the muscle to the lateral border of the latissimus dorsi in preoperative localization and intraoperative exploration, the diameter of thoracodorsal artery perforator measured during operation, and the flap types were recorded. The survivals of flaps and appearances of donor sites were followed up. Results: The number and location of thoracodorsal artery perforators located before operation in each patient were consistent with the results of intraoperative exploration. A total of 42 perforators were found in two trapezoidal areas, with 2 or 3 perforators each patient. The perforators were all located in two trapezoid areas, and a stable perforator (the first perforator) was located and detected in the first trapezoidal area. There were averagely 1.47 perforators in the second trapezoidal area. The position where the first perforator exits the muscle was 2.1-3.1 cm away from the lateral border of the latissimus dorsi. The diameters of thoracodorsal artery perforators were 0.4-0.6 mm. In this group, 12 cases were repaired with single thoracodorsal artery perforator flap, 3 cases with lobulated thoracodorsal artery perforator flap, and 2 cases with thoracodorsal artery perforator flap carrying partial latissimus dorsi muscle. The patients were followed up for 6 to 16 months. All the 17 flaps survived with good elasticity, blood circulation, and soft texture. Only linear scar was left in the donor area. Conclusions: The two longitudes three transverses method is helpful to locate the perforator of thoracodorsal artery perforator flap. The method is simple and reliable. The thoracodorsal artery perforator flap designed and harvested based on this method has good clinical effects in repairing deep wound, with minimal donor site damage.

[MicroRNA 424-5p promotes the sensitivity of hepatocellular carcinoma cells to sorafenib by targeting Kinesin family member 23].

Objective: To explore the effect of MicroRNA 424-5p/Kinesin family member 23(miR-424-5p/KIF23)axis on the malignant phenotype of hepatoma cells and its sensitivity of sorafenib. Methods: Real-time quantitative reverse PCR(qRT-PCR) and/or Western blot were used to detect the expression of miR-424-5p and KIF23 in liver cancer tissues and paracancerous tissues, human hepatocellular carcinoma(HCC) cells HepG2 and normal hepatocyte LO2. HepG2 cells transfected with miR-424-5p mimic and miR-424-5p mimic NC were respectively defined as miR-424-5p mimic group and mimic NC group, HepG2 cells transfected with KIF23 overexpression vector pcDNA3.1-KIF23 or empty vector pcDNA3.1 respectively were defined as OE-KIF23 group and Vector group, and HepG2 cells co-transfected with miR-424-5p mimic and overexpression vector pcDNA3.1-KIF23 were defined as mimic+OE-KIF23 group: The KIF23-3'UTR wild-type vector (KIF23-WT) and the mutant vector (KIF23-MT) were co-transfected with miR-424-5p micic and mimic NC, respectively, and the targeting relationship between miR-424-5p and KIF23 was verified by dual-luciferase reporting experiments. The cell counting Kit-8 (CCK-8) was used to detect HepG2 cell proliferation and sensitivity to sorafenib. Flow cytometry was used to assess apoptosis in HepG2 cells. Transwell and scratch experiments were used to detect HepG2 cell migration and invasion capabilities. Intergroup data were compared using t-tests or analysis of variance. Results: Compared with the paracancerous tissue and normal hepatocytes, miR-424-5p in the HCC tissue and hepatocellular cells was significantly down-regulated (the relative expression was 0.604±0.121, 0.585±0.064), and KIF23 was significantly up-regulated (the relative expression was 5.451±1.834, 2.482±0.545), P<0.05. miR-424-5p mimic can inhibit the proliferation, migration and invasion of HCC cells and promote apoptosis of HCC cells (P<0.05). Overexpression of KIF23 can promote the proliferation, migration and invasion of HCC cells and inhibit apoptosis of HCC cells (P<0.05). The luciferase activity of HepG2 cells in the mimic and KIF23-WT co-transfection groups was significantly reduced compared with HepG2 cells in the mimic NC and KIF23-WT co-transfection groups (the relative fluorescence intensities were 3.668±0.091 and 2.629±0.056, respectively, P<0.05),however, there was no significant comparison between the luciferase activity of cells in the mimic and KIF23-MT co-transfection groups compared with those in the mimic NC and KIF23-MT co-transfection groups. miR-424-5p mimic can reverse the role of overexpression of KIF23 in promoting the ability of HCC cells to proliferate, migrate and invade (P<0.05). The inhibition rates of sorafenib on HepG2 cells in the mimic+OE-KIF23 group and the OE-KIF23 group were 47.491%±3.863% and 36.246%±6.063% (t=3.027, P<0.05). Conclusion: miR-424-5p can inhibit the proliferation, migration and invasion of HCC cells and can increase the sensitivity of HCC cells to sorafenib by targeting the expression level of KIF23.

[Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice].

Objective: To investigate the feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice. Methods: (1) Ten eight-week-old C57BL/6 male mice were collected and full-thickness skin tissue with diameter of 1.0 cm on both sides of the midline of the back was taken with a perforator to make the normal skin tissue homogenate supernatant. At 48 h after the full-thickness skin defect wound was established, the wound tissue within 2 mm from the wound edge was taken to make inflammatory wound tissue homogenate supernatant. Two kinds of tissue homogenate supernatant were taken to adjust the total protein concentration to 1 mg/mL, and the tumor necrosis factor α (TNF-α) content was detected by enzyme-linked immunosorbent assay. The number of sample was 6. (2) The primary passage of human umbilical cord mesenchymal stem cells (hUCMSCs) were collected and cultured to the 3rd passage with the normal exosomes being extracted from the hUCMSCs after cultured for 48 h. Another batch of hUCMSCs in the 3rd passage was collected and stimulated with inflammatory wound tissue homogenate supernatant of 30, 50, and 100 µg/mL total protein and normal skin tissue homogenate supernatant of 30, 50, and 100 µg/mL total protein, respectively. After cultured for 48 h, the exosomes stimulated with normal protein of 30, 50, and 100 µg/mL and exosomes stimulated with inflammatory protein of 30, 50, and 100 µg/mL were extracted. Normal exosomes, exosomes stimulated with 30 µg/mL normal protein, and exosomes stimulated with 30 µg/mL inflammatory protein were collected, the morphology was observed by transmission electron microscope, the particle size was detected by nanoparticle tracking analyzer, and the expressions of CD9 and CD63 were detected by Western blotting. (3) Twenty one-day-old C57BL/6 mice were taken to isolate the primary passage of fibroblasts (Fbs) and the 3rd passage of Fbs, whose morphology was observed under the inverted phase contrast microscope. The Fbs of 3rd passage were collected to observe the expression of vimentin by cell crawling method combined with immunofluorescence method at culture hour (CH) 2. (4) The Fbs of 3rd passage were divided into control group, normal exosome group, 30, 50, 100 µg/mL normal protein stimulating exosome group, and 30, 50, 100 µg/mL inflammatory protein stimulating exosome group according to the random number table, with 4 wells in each group. Cells in control group received no treatment, and cells in the other 7 groups were respectively added with normal exosomes, exosomes stimulated with normal protein of 30, 50, and 100 µg/mL, and exosomes stimulated with inflammatory protein of 30, 50, and 100 µg/mL prepared in experiment (2). The final mass concentration of exosomes was adjusted to 10 µg/mL. The cell viability was detected by cell count kit 8 at CH 48. (5) Two batches of Fbs in the 3rd passage were divided and treated as those in experiment (4), with 4 wells in each group, and the final mass concentration of exosomes was adjusted to 1 and 10 µg/mL, respectively. The cell mobility was detected by cell scratch test at CH 6, 12, and 24. (6) Two batches of the Fbs of 3rd passage were collected, divided, and treated as those in experiment (4) except with no control group, with 3 wells in each group, and the final mass concentration of exosomes was respectively adjusted to 1 and 10 µg/mL. The mRNA expression levels of transforming growth factor ß(1) (TGF-ß(1)), TGF-ß(3), and α smooth muscle actin (α-SMA) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction at CH 48. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Bonferroni method. Results: (1) The content of TNF-α in inflammatory wound tissue homogenate supernatant of mice was (116±3) pg/mL, significantly higher than (97±5) pg/mL in normal skin tissue homogenate supernatant at post injury hour 48 (t=3.306, P<0.05). (2) Normal exosomes, exosomes stimulated with 30 µg/mL normal protein, and exosomes stimulated with 30 µg/mL inflammatory protein of hUCMSCs showed the typical saucer-like shape. The particle sizes of the three exosomes of hUCMSCs were 30-150 nm, which were all within the normal particle size range of exosome. Three exosomes of hUCMSCs positively expressed CD9 and CD63. (3) The primary passage of cells were clearly defined and showed protruding spindle shape, irregular polygon shape, or slender strip shape. The morphology of the 3rd and the primary passage of cells is similar. At CH 2, vimentin in cells was positively expressed, and the cells were identified as Fbs. (4) At CH 48, the cell viability was (137.4±2.8)% in 30 µg/mL inflammatory protein stimulating exosome group, obviously higher than 100%, (107.5±2.4)%, (113.3±3.2)%, (104.0±2.0)%, and (101.9±1.5)% in control group, normal exosome group, 30 µg/mL normal protein stimulating exosome group, and 50 and 100 µg/mL inflammatory protein stimulating exosome groups, respectively (P<0.01), and cell viability in 30 µg/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 µg/mL normal protein stimulating exosome groups [(103.4±2.2)% and (102.5±1.4)%], respectively (P<0.01). (5) At CH 6, 12, and 24, the mobility rate of cells in 30 µg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, 30 µg/mL normal protein stimulating exosome group, and 50 and 100 µg/mL inflammatory protein stimulating exosome groups, respectively, when the final mass concentrations of exosome was 1 µg/mL (P<0.05) . At CH 12, the mobility rate of cells in 30 µg/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 µg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 1 µg/mL (P<0.05). At CH 6, the mobility rate of cells in 30 µg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group and normal exosome group (P<0.05), and the mobility rate of cells in 30 µg/mL normal protein stimulating exosome group was significantly higher than that in 50 and 100 µg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 µg/mL (P<0.05). At CH 12 and 24, the mobility rate of cells in 30 µg/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 µg/mL inflammatory protein stimulating exosome groups (P<0.05), and the mobility rate of cells in 30 µg/mL normal protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 µg/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 µg/mL (P<0.05). (6) There were no statistically significant differences in mRNA expression levels of TGF-ß(1), TGF-ß(3), and α-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 1 µg/mL (F=1.123, 1.537, 1.653, P>0.05). There were no statistically significant differences in mRNA expression levels of TGF-ß(1) and α-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 10 µg/mL (F=1.487, 1.308, P>0.05), and mRNA expression level of TGF-ß(3) of cells in 50 µg/mL inflammatory protein stimulating exosome group at CH 48 was significantly higher than that in normal exosome group, 50 µg/mL normal protein stimulating exosome group, and 30 and 100 µg/mL inflammatory protein stimulating exosome groups when the final mass concentration of exosome was 10 µg/mL (P<0.05). Conclusions: The pretreatment with inflammatory wound tissue homogenate supernatant of mice has no significant effect on the total protein of hUCMSCs exosomes. The hUCMSCs exosomes stimulated by low concentration inflammatory wound tissue homogenate supernatant can significantly promote the proliferation and migration ability of Fbs. The content of inflammatory mediators in the wound tissue homogenate supernatant during the inflammatory phase is extremely low, which may be the reason that the anti-inflammation and tissue repair paracrine effects of mesenchymal stem cell cannot be effectively started.

[Clinical effects of modified fascia flap from cutaneous branch of dorsal metacarpal artery in repairing the wound at the proximal and middle finger segments].

Objective: To investigate the clinical effects of modified fascia flap from cutaneous branch of dorsal metacarpal artery in repairing the wound at the proximal and middle finger segments. Methods: From January 2017 to September 2018, 12 patients with wounds at the proximal and middle finger segments were admitted to the Affiliated Hospital of Zunyi Medical University, including 8 males and 4 females, aged 35-70 years. The areas of wounds ranged from 3.4 cm×2.4 cm to 6.5 cm×4.0 cm. The modified fascia flaps from cutaneous branch of dorsal metacarpal artery were resected to repair the wounds, with the size ranging from 3.5 cm×2.5 cm to 6.7 cm×4.1 cm. The flap donor sites of 5 patients were repaired with direct intermittent suture, the flap donor sites of 4 patients were repaired with full-thickness skin grafts from ipsilateral medial forearm, and the flap donor sites of 3 patients were repaired with wrist pedicled flaps. The survival of the flaps was recorded. Healing of donor site and recipient site was followed. The hand functions were evaluated with trial standard for the evaluation of the functions of the upper limbs of the Hand Surgery Society of the Chinese Medical Association. Results: All the flaps survived in 12 cases. During 3 to 12 months of follow-up, the flaps recovered satisfactorily in texture and shape. The donor sites of 11 patients were healed, and the skin graft edge area was partially necrotic in the other patient but healed later after dressing change. The distances of two-point discrimination of the patients ranged from 5.6 to 9.0 mm. Hand functions were evaluated as excellent in 5 cases, good in 4 cases, and fair in 3 cases. Conclusions: Modified fascia flap from cutaneous branch of dorsal metacarpal artery for repairing the wounds at the proximal and middle finger segments has reliable blood supply. The operation is simple and safe with short course of treatment, which is worthy of clinical promotion.

[Research update of effects of adipose tissue and component transplantation on scar treatment].

Scar is a kind of skin fibroproliferative disease characterized by excessive repair of skin tissue and disorganized deposition of extracellular matrix resulting from deep dermal injury caused by burns or trauma. Scar is accompanied by symptoms such as itching and pain, which could lead to appearance damage and psychological disorders, and is one of the common diseases in burns and plastic surgery clinics. Currently, transplantation of adipose tissue and components is considered as one of the most cutting-edge treatments for scar. Adipose components transplantation includes transplantation of nanofat, adipose-derived stem cell matrix gel, stromal vascular fraction, and adipose-derived stem cell. More and more studies showed that adipose tissue and components possess the functions of tissue regeneration, extracellular matrix remodeling, and anti-fibrosis, which could improve the appearance and symptoms of scar by local transplantation. Therefore, this paper reviews the effects of adipose tissue and components transplantation on scar treatment, aiming to provide theoretical reference for adipose treatment of scar.

[Advances in the research of effects of epithelial-mesenchymal interaction on wound healing and scar formation].

Wound healing is a dynamic process which involves interaction of various types of cells, cytokines, and extracellular matrix. Among them, epithelial cells and mesenchymal cells are the key components which involve in wound healing and scar formation. Related scholars had done a great number of studies about the functions of epithelial cells and fibroblasts(Fbs) in wound healing and scar formation. The results showed that under the stimulation of complex microenvironment, epithelial cells would lose their epithelial characteristics and acquire the typical characteristics and migration ability of mesenchymal cells. At the same time, with the complex changes of cell structure and cell behavior, they would participate in the process of tissue wound repair, including normal or fibrotic repair, by covering the wound with migration. Fbs are the key cells for the wound fibrotic repair, and play important roles in the process of wound healing, including excessive wound healing or delayed wound healing. In the recent years, the researchers realized that the cross-talk between epithelial cells and Fbs in wound healing, which is referred to as epithelial-mesenchymal interaction, significantly changes the biological behaviors of these two cell types, which affects the dermal remodeling and re-epithelialization quality of wound. Epithelial-mesenchymal interaction plays an important role in skin morphogenesis during embryonic development and maintaining the structural integrity of adult skin. In the process of re-epithelialization, Fbs could promote the proliferation and migration of keratinocytes, meanwhile keratinocytes would receive the signals from Fbs to reconstruct functional epithelium, which has become a hot topic in the field of wound healing at present. In this paper, a comprehensive analysis of the literature on the role of epithelial-mesenchymal interaction in wound healing and scar formation at home and abroad in recent years is presented for the reference of relevant scholars.

[Influence of human amniotic mesenchymal stem cells on macrophage phenotypes and inflammatory factors in full-thickness skin wounds of mice].

Objective: To explore the influence of human amniotic mesenchymal stem cells (hAMSCs) on the in vivo and in vitro regulation of macrophage phenotypes and inflammatory factors associated with wound healing of full-thickness skin wounds in mice. Methods: Fresh amniotic membrane discarded from full-term delivery by 5 healthy pregnant women in the Department of Obstetrics and Gynecology of the Affiliated Hospital of Zunyi Medical University was used for the isolation and culture of hAMSCs by enzyme digestion method. The third passage of cells was used for identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of hAMSCs surface markers. Ten C57BL/6 mice (all male, aged 6 to 8 weeks, the same gender and age below) were selected for extracting mouse peritoneal macrophages by intraperitoneal lavage, and M1-type macrophages were induced by Dulbecco's modified eagle medium (DMEM) medium containing interferon-γ. The M1-type macrophages were divided into hAMSCs+ macrophage group and macrophage alone group. Then 1×10(4) hAMSCs/per well of fourth passage were added to macrophage in hAMSCs+ macrophage group and cultured in 2 mL DMEM medium for routine culture. In macrophage alone group, each well was only added with 2 mL DMEM medium for routine culture. On day 1 and 7 in culture, the content of interleukin-12 (IL-12), arginase 1, and IL-10 in the cell culture supernatant of the 2 groups were detected by enzyme-linked immunosorbent assay with sample number of 6/per group. (2) Full-thickness skin wound model was reproduced in the back of 56 C57BL/6 mice, which were divided into hAMSCs group and phosphate buffer solution (PBS) group using the random number table, with 28 mice in each group. Mice in hAMSCs group were subcutaneously injected with 100 µL of cell suspension containing 1×10(7) hAMSCs per mL in PBS suspension along the wound edge. While mice in PBS group were only subcutaneously injected with 100 µL PBS along the wound edge. On post injection day (PID) 1, 3, 7, and 14, 7 mice in the two groups were sacrificed respectively. Histopathological observation was performed with hematoxylin-eosin staining. The expressions of macrophage surface markers [CD68 and inducible nitric oxide synthase (iNOS) double positive cells and CD68 and arginase 1 double positive] in the wounds were detected by immunofluorescent staining. The mRNA expressions of IL-10, macrophage inflammatory protein 1α (MIP-1α), and MIP-2 in the wounds were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with analysis of variance for factorial design, t test, and Bonferroni correction. Results: (1) On day 1 in culture, the content of IL-12 and arginase 1 in the cell culture supernatant of the two groups were similar (t=0.448, 0.536, P>0.05), and the content of IL-10 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group (t=14.722, P<0.01). On day 7 in culture, the content of IL-12 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group (t=13.226, P<0.01), and the content of arginase 1 and IL-10 was significantly higher than that in macrophage alone group (t=30.172, 31.406, P<0.01). (2) On PID 1, a large number of inflammatory cells infiltration were observed in the skin wounds of both groups. On PID 3, the inflammatory cells infiltration in the skin wounds increased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 7, the inflammatory cells infiltration in the wounds decreased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 14, no obvious inflammatory cells infiltration was observed in the wounds in the two groups. (3) On PID 1 and 14, the percentages of CD68 and iNOS double positive cells and CD68 and arginase 1 double positive cells in the wounds were similar in the two groups (t(1 d)=0.134, 0.693, t(14 d)=1.146, 2.585, P>0.05). On PID 3 and 7, the percentages of CD68 and iNOS double positive cells in the wounds in hAMSCs group were significantly lower than those of PBS group (t=6.396, 4.787, P<0.01), while the percentages of CD68 and arginase 1 double positive cells were significantly higher than those of PBS group (t=3.928, 4.473, P<0.01). (4) On PID 1, the mRNA expressions of IL-10 in the wounds of mice in the two groups were similar (t=2.005, P>0.05). On PID 3, 7, and 14, the mRNA expressions of IL-10 in the wounds of mice in hAMSCs group were significantly higher than those of PBS group (t=7.758, 124.355, 80.823, P<0.01). On PID 1, 3, 7, and 14, the mRNA expressions of MIP-1α and MIP-2 in the wounds of mice in hAMSCs group (0.341±0.212, 0.648±0.004, 0.611±0.106, 0.763±0.049, 1.377±0.099, 1.841±0.042, 1.181±0.035, 0.553±0.028) were significantly lower than those of PBS group (3.853±0.035, 6.914±0.163, 3.648±0.113, 2.250±0.046, 11.119±0.495, 8.634±0.092, 5.722±0.021, 4.862±0.036, t=43.198, 101.904, 51.845, 58.231, 51.074, 177.501, 291.752, 251.614, P<0.01). Conclusions: hAMSCs demonstrates biological effects of promoting the transformation of M1-type macrophages into M2-type macrophages in full-thickness skin wounds of mice. They can up-regulate the expression of anti-inflammatory and anti-fibrotic factor IL-10, and down-regulate the expression of important inflammation mediated factors MIP-1α and MIP-2.

[Effects of adipose-derived mesenchymal stem cells from type 2 diabetes mellitus patients on wound healing of pressure ulcers in mice].

To investigate the effects of adipose-derived mesenchymal stem cells (AMSCs) from type 2 diabetes mellitus patients on wound healing of pressure ulcers in mice. Methods: (1) In September 2016, the subcutaneous adipose tissue of a 60-year-old woman with type 2 diabetes mellitus was harvested, and then AMSCs were extracted by collagenase digestion and cultured. The third passage of cells were used for subsequent experiments. The morphology of cells was observed, and their osteogenic, chondrogenic, and adipogenic differentiation abilities were identified. The expressions of cell surface markers CD90, CD105, CD73, and CD34 were detected by flow cytometer (n=3). (2) Sixteen female C57BL/6 wild-type mice aged 6-8 weeks were selected, and one pressure ulcer wound was created on each side of the spine of each mouse by pressing the skin with two magnets. The two wounds of each mouse were paired and divided into diabetic AMSCs group and negative control group, injected with 100 µL phosphate buffer solution (PBS) containing green fluorescent protein-labeled AMSCs (1×10(6) cells) and 100 µL PBS, respectively. The wound healing status of the two groups within post injection day (PID) 21 was observed, and their wound healing rates on PID 5, 13, and 17 were calculated. Three mice were sacrificed on PID 11 and 21, respectively, and tissue of three wounds was harvested from each group. The skin structure was observed by hematoxylin-eosin staining, the collagen deposition was evaluated by Masson staining, and the positive expression of CD31, i. e., the number of new blood vessels was counted by immunohistochemistry. Wound tissue samples of two groups prepared on PID 21 as above-mentioned were harvested, and the positive cell rate of S100, representing the regeneration of Schwann cells, was detected by immunohistochemistry. Wound tissue samples of diabetic AMSCs group prepared on PID 11 as above-mentioned were harvested, and the colonization of AMSCs was observed by fluorescence tracer method. Data were processed with paired t test and Bonferroni correction. Results: (1) The third passage of cells isolated and cultured from the subcutaneous adipose tissue of a type 2 diabetes mellitus patient grew adherently to the wall in a long spindle and vortex-like manner. After induction, the cells showed osteogenic, chondrogenic, and lipogenic differentiation abilities. The positive expression rates of CD90, CD105, and CD73 on the cell surface were higher than 90.00%, and the expression rate of CD34 was 0.46%. The cells were identified as AMSCs. (2) The mice wounds of diabetic AMSCs group healed quickly, and all the wounds healed completely on PID 17, while the mice wounds in negative control group were not completely closed at this time, and there was still scab on the surface. On PID 5, 13, and 17, the healing rates of mice wounds of diabetic AMSCs group were (35.6±6.5)%, (87.1±2.5)%, and 100.0%, respectively, significantly higher than (19.8±7.2)%, (66.2±5.2)%, and (86.9±5.3)% of negative control group (t=6.49, 14.31, 9.73, P<0.05). Compared with that of negative control group, the inflammatory cell infiltration was reduced in mice wounds tissue of diabetic AMSCs group on PID 11, and thicker epidermis and dermis as well as regenerated skin appendages were observed on PID 21. On PID 11 and 21, the collagen percentages of mice wounds tissue in diabetic AMSCs group was (48.3±1.3)% and (54.1±1.7)%, respectively, significantly higher than (41.4±1.7)% and (50.3±1.2)% of negative control group (t=6.98, 3.99, P<0.01). On PID 11 and 21, the numbers of new blood vessels in mice wounds tissue of diabetic AMSCs group were 17.2±1.3 and 18.0±2.1, respectively, significantly more than 8.0±1.4 and 14.0±1.5 of negative control group (t=10.69, 3.38, P<0.01). On PID 21, the S100 positive cell percentage in mice wounds tissue of diabetic AMSCs group was (1.76±0.12)%, significantly higher than (0.55±0.03)% of negative control group (t=21.68, P<0.001). On PID 11, the colonization of AMSCs in mice wounds tissue of diabetic AMSCs group was observed. Conclusions: Transplantation of AMSCs from type 2 diabetic mellitus patients can accelerate wound healing of pressure ulcers in mice by promoting angiogenesis, collagen deposition, and Schwann cell regeneration.

[Advances in the research of influence of diabetes in biological function of adipose-derived stem cells].

Adipose-derived stem cells (ADSCs) are adult mesenchymal stem cells in adipose tissue with self-renewal and multi-directional differentiation potential. The application of ADSCs in the treatment of wounds has achieved good results. Because of its extensive sources, high content in vivo, low immunogenicity, slight injury to body when obtained, the clinical application prospect of ADSCs is promising. The reasons why diabetic wound is difficult to heal may be closely related to the increase of advanced glycation end products, long-term chronic inflammatory response, and peripheral neurologic dysfunction. The abnormal internal environment of diabetic patients can affect the biological function of ADSCs, which further affects wound healing. This article reviews the general feature, differentiation, proliferation, migration, secretion, and pro-angiogenic function of diabetic ADSCs.

[Effects of local transplantation of autologous adipose-derived stromal vascular fraction on the hyperplastic scar formation in rabbit ears and the mechanism].

Objective: To explore the effects of local transplantation of autologous adipose-derived stromal vascular fraction (SVF) on the hyperplastic scar (HS) formation in rabbit ears and the mechanism. Methods: Twenty-four New Zealand white rabbits were used to reproduce HSs by making four full-thickness skin defect wounds with a diameter of 1 cm on the ventral surface of left ear of each rabbit. Wound epithelization and local-tissue proliferation were observed, and wound healing (complete epithelization) time and formation time of HS were recorded. The 24 rabbits were divided into SVF group, pure DMEM group, and pure HS group according to the random number table, with 8 rabbits and 32 wounds in each group. On post injury day (PID) 25 (after the complete epithelization of wounds), 0.2 mL of low glucose DMEM medium containing CM-Dil labeled autologous SVF was injected into HSs of rabbits in SVF group, while the same amount of low glucose DMEM medium was injected into HSs of rabbits in pure DMEM group. The frequency of injection was once every 5 days, totally for 3 times. HSs of rabbits in pure HS group did not receive any treatment. On PID 40, HSs of rabbits' ears in each group were harvested, then the histological form was observed by hematoxylin and eosin staining, the arrangement of collagen in HS was observed by Van Gieson staining, the distribution of CM-Dil-labeled SVF in the HS was observed with fluorescence microscope, and the mRNA expression and the protein expression of transforming growth factor ß(1) (TGF-ß(1)), Smad3, and Smad7 in HS were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. Data were processed with one-way analysis of variance and Tukey test. Results: (1) Complete epithelization time of wounds of rabbits' ears was (20.0±2.0) d post injury, and HSs were formed on PID 25. On PID 40, HSs of rabbits' ears in pure DMEM group and pure HS group were still in hyperplasia, while those in SVF group became smaller, flat, soft, and light colored. (2) On PID 40, compared with those in pure DMEM group and pure HS group, the number of epithelium foot like structures was more and the amount of inflammatory cells was less. The collagen of HSs of rabbits' ears in SVF group was arranged more regularly with broader gap between collagens. (3) On PID 40, CM-Dil-labeled SVF could still be observed in the HSs of rabbits' ears in SVF group. (4) On PID 40, compared with those in pure DMEM group and pure HS group, the mRNA expressions of TGF-ß(1) and Smad3 in the HSs of rabbits' ears in SVF group were significantly down-regulated (P<0.05), while the mRNA expression of Smad7 was significantly up-regulated (P<0.05). There were no significant differences in the mRNA expressions of TGF-ß(1), Smad3, and Smad7 in the HSs of rabbits' ears between pure DMEM group and pure HS group (P>0.05). (5) On PID 40, compared with those in pure DMEM group (0.74±0.03, 0.73±0.10, 0.54±0.09) and pure HS group (0.72±0.08, 0.71±0.12, 0.53±0.06), the protein expressions of TGF-ß(1) and Smad3 in the HSs of rabbits' ears in SVF group (0.57±0.06, 0.42±0.09) were significantly down-regulated (P<0.05), while the protein expression of Smad7 (0.71±0.05) was significantly up-regulated (P<0.05). The protein expressions of TGF-ß(1), Smad3, and Smad7 in the HSs of rabbits' ears in pure DMEM group and pure HS group were close (P>0.05). Conclusions: Autologous SVF transplantation can inhibit the formation of HS in the early stage of scar formation of rabbit, the mechanism may be related to the TGF-ß(1)/Smad signaling pathway.

Induction of proliferation and activation of rat hepatic stellate cells via high glucose and high insulin.

OBJECTIVE: To further investigate the occurrence mechanism of diabetic hepatic fibrosis through observing the effects of insulin and glucose in different concentrations on hepatic stellate cell (HSC) proliferation, and mRNA expressions of transforming growth factor-ß1 (TGF-ß1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in rats, so as to provide a theoretical and experimental basis for the occurrence, prevention and treatment of diabetic hepatic fibrosis (HF). MATERIALS AND METHODS: The HSCs in rats were cultured in vitro with high glucose alone and high glucose + high insulin as the stimulating factors and mannitol as the high osmotic pressure control. After the above 10 groups of HSC were cultured for some time, the absorbance value of each group was determined using the Cell Counting Kit-8 (CCK-8) to clarify the number of proliferative HSC. Moreover, the count per minute (Cpm) of DNA in HSC was detected via the 3H-thymidine incorporation (3H-TDR incorporation) to clear the proliferation status of HSC. Finally, the mRNA expressions of TGF-ß1 and TIMP-1 in HSC in each group were detected via Real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR). RESULTS: Both HSC proliferation and DNA synthesis were increased in a glucose concentration-dependent manner, while the HSC proliferation and DNA synthesis in glucose groups with insulin were significantly higher than those in glucose groups without insulin (p<0.05). The DNA synthesis in insulin + mannitol group was higher than that in insulin + normal glucose group. The mRNA level in TGF-ß1 in glucose groups with insulin was decreased, but that in TIMP-1 was increased. CONCLUSIONS: Both high glucose and high insulin can induce the HSC proliferation, and high insulin can further activate HSC and promote the progression of hepatic fibrosis course.

[Clinical effects of middle and low peroneal artery perforator flap with pedicle on repairing skin and soft tissue defects of ankle].

Objective: To investigate clinical effects of middle and low peroneal artery perforator flap with pedicle on repairing skin and soft tissue defects of ankle. Methods: Twenty patients with skin and soft tissue defects of ankle and exposure of tendon and bone were admitted in our burn wards from April 2012 to December 2015. The size of skin and soft tissue defects ranged from 5 cm×4 cm to 23 cm×10 cm. Patients were treated with debridement and vacuum sealing drainage (VSD) after admission. After VSD treatment for 1 week, flap transplantation operation was performed. Middle and low perforating branches of peroneal artery were detected by portable Doppler blood flow meter before the operation. Flaps were designed and resected according wounds during the operation, with 1 or 2 middle and low perforating branches of peroneal artery in flaps. Seventeen patients were treated with middle and low peroneal artery perforator flap. Larger wounds with exposure of tendon and bone were repaired with middle and low peroneal artery perforator flap, and the other wounds were repaired with intermediate split-thickness skin graft of thigh on the same side in three patients. The size of flap ranged from 6 cm×5 cm to 25 cm×12 cm. The donor sites were sutured directly or repaired with intermediate split-thickness skin graft of thigh on the same side. Results: After operation, 1 patient with partial skin necrosis at the distal of the flap because of disorder of venous circulation healed after dressing change and physiotherapy, and flaps of the other 19 patients survived well. During follow-up of 3 to 36 months, flaps of all patients were in good appearance, with no obvious cicatrix, and the affected limbs and ankle joints functioned well. Conclusions: Middle and low peroneal artery perforator flap with advantages of stable perforating branch, reliable blood supply, and large resected size, can repair skin and soft tissue defects of ankle.

SRAP analysis of DNA base sequence changes in lotus mutants induced by Fe⁺ implantation.

Ion implantation, a new biophysically mutagenic technique, has shown great potential for crop breeding. To reveal the mutation effect of low-energy ion implantation on Baiyangdian red lotus, sequence-related amplified polymorphism markers were used to amplify and detect the DNA sequence differences in mutants induced by Fe(+) ion implantation. A total of 121 primer combinations were tested in 6 mutants and a control. Seven primer combinations (me1 + em3, me1 + em14, me9 + em3, me8 + em2, me6 + em1, me11 + em5, and me6 + em5) generated clear bands with high polymorphism and good repeatability. The results showed that among 15,317 bases cloned, 146 bases in 6 mutants were different from those of the wild type, showing a variation frequency of 0.95%. The types of base changes included deletion, insertion, transversion, and transition. Adenine was more sensitive to the irradiation than were the other bases. The results suggested that mutational "hotspots" probably exists in lotus and are induced by low-energy ion implantation.

First Report of Cherry necrotic rusty mottle virus on Stone Fruit Trees in China.

During the growing seasons of 2010 through 2012, leaf tissues from 206 stone fruit trees, including one flowering cherry, three sour cherry, six nectarine (Prunus persica L. var. nucipersica Schneider), 14 apricot, 24 plum (P. domestica L.), 41 sweet cherry, and 117 peach [P. persica (L.) Batsch] trees, grown in six provinces of China, were randomly collected and tested for the CNRMV infection by RT-PCR. Out of those sampled trees, 37 showed shot holes and vein yellowing symptoms. Total RNA was extracted from leaves using the CTAB protocol reported by Li et al. (2). The primer pair CGRMV1/CGRMV2 (1) was used to amplify a fragment of 949 bp from CNRMV genome, which includes the CP gene (804 bp). PCR products with the expected size were detected in one sweet cherry, one apricot, one peach, one plum, and two sour cherry plants. However, no correlation between PCR data and symptom expression could be found. PCR products were cloned into the vector pMD18-T (TaKaRa, Dalian, China). Three independent clones from each isolate were sequenced by Genscript Corp., Nanjing, China, and sequences were deposited in the GenBank under accession nos. JX491635, JX491636, JX491637, JX648205, and JX648206. Results of sequence analysis showed that sequences of the five CNRMV isolates shared the highest nt (99.0 to 99.6%) and aa (98.9 to 100%) similarities with a cherry isolate from Germany (GenBank Accession No. AF237816). The sequence of one isolate from a peach tree (JX648205) was divergent and shared only 84.7 to 86.1% nt and 94.4 to 95.1% aa similarities with those cp sequences. Clones intra each isolate shared more than 99% nt similarities. To confirm CNRMV infection, seedlings of peach GF 305 were graft-inoculated with bud-woods from a peach and a sweet cherry tree, which was positive to CNRMV and also two other viruses: Cherry green ring mottle virus (CGRMV) and Plum bark necrosis stem pitting-associated virus (PBNSPaV), as tested by RT-PCR. Grafted seedlings were kept in an insectproof greenhouse and observed for symptom development. In May of the following year, some newly developed leaves of inoculated seedlings showed vein yellowing, ringspot, and shot hole symptoms. Results of Protein A sandwich (PAS)-ELISA using an antiserum raised against the recombinant CP of a CNRMV isolate (unpublished) and RT-PCR confirmed CNRMV infection in inoculated trees. In addition to CNRMV, tested seedlings were also found to be infected with CGRMV and PBNSPaV by RT-PCR. To our knowledge, this is the first report on the occurrence of CNRMV on stone fruit trees in China, and also the first record of the CNRMV infection in peach and plum plants. Given the economic importance of its hosts and the visible symptoms of the viral disease, it is important to prevent the virus spread by using virus-tested propagation materials. References: (1) R. Li and R. Mock. J. Virol. Methods 129:162, 2005. (2) R. Li et al. J. Virol. Methods 154:48, 2008.

The point mutation induced by the low-energy N+ ion implantation in impatiens balsamine genome.

To investigate the effect of genome mutations induced by low energy ions implantation in higher plants, genome mutation of Impatiens balsamine mutant induced by low energy N+ ion implantation were analyzed by the RAPD, ISSR and genome sequence. Six out of the 121 ISSR primers and 6 out of the 135 RAPD primers showed that polymorphism ratios between mutants and wild type were 4.96% and 2.89%, respectively. Sequence analysis revealed that base deletions, insertions, and substitutions were observed in the mutant genome comparable to wild type. N+ induced point mutations were mostly base substitution (77.4%), no duplication, long fragments insertions and deletions was found. In all point mutation, adenine (A) was most sensitive to the N+ ion implantation in impatiens. The transition was mainly A --> guanine (G) (15.90%) and thymine (T) --> cytosine (C) (12.55%). Transversion happened in A <--> T (16.74%), which much higher than C <--> G(5.02%), G <--> T(6.69%), A <--> C (7.11%) bases. These findings indicate that low energy ions being a useful mutagen were mostly cause the point mutation in impatiens.