Three-dimensional measurement and analysis of botulinum toxin A injection for improving the aesthetic appearance of upper lip.

This study evaluated the efficacy of botulinum toxin A (BTX A) in improving the aesthetic appearance of lips. Twenty-four outpatients with clinical evidence indicating decreased fullness of lips or gummy smile were selected and received BTX A injection on the orbicularis oris. We observed a significant decrease in wrinkles and improvement in gummy smile in all patients 4 weeks after the injection. Aesthetic lines also changed significantly. Local injection of BTX A on the orbicularis oris could simultaneously achieve mild lip enhancement, improvement in fine wrinkles around lips, and mild gummy smile.

Human umbilical cord mesenchymal stem cells implantation accelerates cutaneous wound healing in diabetic rats via the Wnt signaling pathway.

OBJECTIVE: Difficulty in wound healing is one common complication of diabetes mellitus. The study explored whether the therapeutic effect of human umbilical cord mesenchymal stem cells (hUCMSCs) on diabetic ulcer wound was enhanced by the activation of the Wnt signaling pathway. METHODS: Rat diabetic model was established by intraperitoneal injection of Streptozotocin (STZ). hUCMSCs were purified and seeded on the collagen-chitosan laser drilling acellular dermal matrix (CCLDADM) scaffold, which was subsequently implanted into the cutaneous wound of normal and diabetic rats, followed by daily injection of Wnt signaling pathway agonist (Wnt3a) or antagonist (sFRP3) at the edge of the scaffold. Wound healing was checked on days 7, 14, and 21, and the fibrous tissue deposition, capillaries, and epidermal regeneration at the wound were examined by hematoxylin-eosin staining. The hUCMSCs-CCLDADM scaffold was cultured in vitro and treated with Wnt3a or sFRP3, followed by evaluation of cell proliferation, cell proliferation rate, survival status, and altered protein levels in the Wnt signaling pathway using BrdU staining, CCK-8 assay, live/dead staining, and Western blotting, respectively. RESULTS: On days 7 and 14 postoperatively, the speed of wound healing was significantly lower in diabetic rats than that in normal control rats. This phenomenon was significantly improved by the activation of the Wnt signaling pathway that also elevated the fibrous protein deposition and the abundance of capillary in the granulation tissue. Conversely, blockade of Wnt signaling slowed the healing of skin wound in diabetic rats. The activation of Wnt signaling pathway promoted the proliferation and differentiation and decreased the apoptosis of hUCMSCs, thereby elevating the number of living hUCMSCs on the CCLDADM scaffold, while the suppression exerted a contrary effect. CONCLUSION: The activation of the Wnt signaling pathway promotes the healing of diabetic skin wound by the regulation of proliferation and differentiation of hUCMSCs on the CCLDADM scaffold.

Clinical application prospect of umbilical cord-derived mesenchymal stem cells on clearance of advanced glycation end products through autophagy on diabetic wound.

Nowadays, wound healing delay due to diabetes is considered to be closely related to the accumulation of advanced glycation end products (AGEs). Although mesenchymal stem cells (MSCs) exhibit positive effects on diabetic wound healing, related mechanisms are still not fully elucidated. It has been reported that MSCs can improve the activity of autophagy in injured tissues, thereby playing an important role in wound healing. The autophagy induced by MSCs may be beneficial to diabetic wound healing via removing AGEs, which provide new ideas for clinical treatment of diabetic wounds with the potential of broad application prospects. In this study, the current research situation and application prospect of umbilical cord-derived MSCs on the clearance of AGEs in diabetic wound were reviewed.

Microencapsulated VEGF gene-modified umbilical cord mesenchymal stromal cells promote the vascularization of tissue-engineered dermis: an experimental study.

BACKGROUND AIMS: Tissue-engineered dermis (TED) is thought to be the best treatment for skin defect wounds; however, lack of vascular structures in these products can cause slow vascularization or even transplant failure. We assessed the therapeutic potential of microencapsulated human umbilical cord mesenchymal stromal cells (hUCMSCs) expressing vascular endothelial growth factor (VEGF) in vascularization of TED. METHODS: hUCMSCs were isolated by means of enzymatic digestion and identified by means of testing biological characteristics. hUCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. Collagen-chitosan laser drilling acellular dermal matrix (ADM) composite scaffold was prepared by means of the freeze dehydration and dehydrothermal cross-linking method. hUCMSC-derived fibroblasts were implanted on composite scaffolds to construct TED. TED with microencapsulated VEGF gene-modified hUCMSCs was then transplanted into skin defect wounds in pigs. The angiogenesis of TED at 1 week and status of wound healing at 3 weeks were observed. RESULTS: The collagen-chitosan laser ADM composite has a uniform microporous structure. This composite has been used to grow hUCMSC-derived fibroblasts in vitro and to successfully construct stem cell-derived TED. Microencapsulated VEGF gene-modified hUCMSCs were prepared with the use of a sodium alginate-barium chloride one-step encapsulation technology. Seven days after the transplantation of the stem cell-derived TED and microencapsulated VEGF gene-modified hUCMSCs into the skin defect wounds on the backs of miniature pigs, the VEGF expression increased and the TED had a higher degree of vascularization. Re-epithelialization of the wound was completed after 3 weeks. CONCLUSIONS: Microencapsulated VEGF gene-modified hUCMSCs can effectively improve the vascularization of TED and consequently the quality of wound healing.

A novel model of burn-blast combined injury and its phasic changes of blood coagulation in rats.

Burn-blast combined injury has a complex pathological process that may cause adverse complications and difficulties in treatment. This study aims to establish a standard animal model of severe burn-blast combined injury in rats and also to investigate early phasic changes of blood coagulation. By using 54 Wistar rats, distance from explosion source (Hexogen) and size of burned body surface area were determined to induce severe burn-blast combined injury. Thereafter, 256 rats were randomly divided into four groups (n = 64): blast injury group, burn injury group, burn-blast combined injury group, and sham injury group. Gross anatomy and pathological changes in lungs were investigated at 3, 24, 72, and 168 h, respectively. Blood was also collected for analyzing coagulation parameters as prothrombin time, activated partial thromboplastin time, and plasma levels of fibrinogen, D-dimer, antithrombin III, and α2-antiplasmin from 0 to 168 h after injury. Severe burn-blast combined injury was induced by inflicting rats with a moderate blast injury when placing rats 75 cm away from explosion source and a full-thickness burn injury of 25% total body surface area. The rats with burn-blast combined injury had more severe lung injuries when compared with the other three groups. Pathological examination in the BBL group showed diffused alveolar hemorrhage, fluid filling, alveolar atelectasis, rupture and hyperplasia of partial alveolar septum, emphysema-like change, reduced capillary bed, and infiltration of extensive polymorphonuclear cells after injury. The blood of combined injured rats was in a hypercoagulable state within 24 h, shortly restored from 24 to 48 h, and rehypercoagulated from 48 to 72 h after injury. A secondary excessively fibrinolytic function was also found thereafter. The rat model of burn-blast combined injury was successfully established by simulating real explosion characteristics. Rats with burn-blast combined injuries suffered from more severe lung injuries and abnormal coagulation and fibrinolytic function than those induced by a burn injury or a blast injury component. Hence, a time-dependent treatment strategy on coagulation function should be emphasized in clinical therapy of burn-blast combined injury.

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro.

Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.

[Effect of different concentration of tamoxifen ointment on the expression of TGF-beta2 of hypertrophic scar at rabbit ears].

OBJECTIVE: To observe the effect of different concentration of Tamoxifen ointment on the fibroblasts and transforming growth factor (TGF-beta2) of hypertrophic scar at rabbit ears, so as to explore the possibility of treatment of hypertrophic scar with Tamoxifen. METHODS: The hypertrophic scar model was established in 96 New Zealand rabbits' ears. The wounds were divided into four groups (A, B, C and D), with 144 wounds in each group. Different concentration of tamoxifen ointment (0.5%, 1%, 2%) was topically administered in groups A, B and C respectively, and blank ointment in group D. On postoperative day 30, 60 and 90, the scar samples were harvested. The scar thickness, scar histological change and the content of TGF-beta2 were detected. RESULTS: (1) On the 30th day after operation, the difference of scar tissue thickness among groups A, D and B, C reached statistical significance (group A, D < group B < group C). However, there was a contrary tendency in fibroblasts density and TGF-beta2 content of the scar tissue simultaneously. (2) On 60th, 90th day after injury, there was statistical difference in scar thickness, fibroblasts density and the content of TGF-beta2 in scar of four groups (P < 0.05). The content of TGF-beta2 in group A, B, C, D was (43.97 +/- 3.63) microg/L, (41.92 +/- 3.91) microg/L, (36.69 +/- 4.15) microg/L, (54.90 +/- 4.71) microg/L, respectively, on 60th day; and (45.69 +/- 2.63) microg/L, (40.43 +/- 3.87) microg/L, (38.76 +/- 3.24) microg/L, (52.59 +/- 4.92) microg/L, respectively, on 90th day. The fibroblasts density of scar in groups A, B, C, D was (4392.07 +/- 327.84) point/mm2, (4208.57 +/- 329.76) point/mm2 (4 033.44 +/- 427.91) point/mm2, (4863.03 +/- 387.98) point/mm2, respectively, on 60th day; and (4418.41 +/- 432.52) point/mm2, (4077.65 +/- 386.70) point/mm2, (3844.53 +/- 354.29) point/mm2, (4838.64 +/- 390.52) point/mm2, respectively, on 90th day. The content of TGF-beta2 and fibroblasts density of scar were lined up as group D > group A > group B > group C (P < 0.05). CONCLUSIONS: Topical Tamoxifen can reduce the content of TGF-beta2 and fibroblast, decrease fibroblasts density and the formation of hypertrophic scar at rabbit ears. It offers a new way for the treatment of the hypertrophic scar.

[Research progress of biological characteristics and advantages of Wharton's jelly-mesenchymal stem cells].

OBJECTIVE: To summarize the research progress of biological characteristics and advantages of Wharton's jelly-mesenchymal stem cells (WJ-MSCs). METHODS: The related literature on the biological characteristics of WJ-MSCs, umbilical cord blood MSCs (UBMSCs) and bone marrow MSCs (BMSCs) was extensively reviewed and analyzed. RESULTS: A large number of MSCs which are able to self-replicate, self-renew and have high proliferation and multipotent differentiation can be isolated from the Wharton's jelly of umbilical cord. WJ-MSCs have many advantages in isolation time, isolation efficiency, expansion time, passage capacity, expansion capacity when compared with UBMSCs and BMSCs. CONCLUSION: WJ-MSCs have numerous advantages of convenient and abundant sources, relatively pure, non-ethical issues, and so on, which can be used for cell transplant therapy, gene therapy, and the ideal seed cells of building tissue engineered organ, so they provide new ideas for tissue regeneration repair and reconstruction.

[Growth and migration of umbilical cord mesenchymal stem cells on polycarbonate membrane with different pore sizes].

OBJECTIVE: To observe the growth and migration of human umbilical cord mesenchymal stem cells (hUCMSCs) on polycarbonate membrane with different pore sizes and explore the criteria of selecting optimal Transwell insert for indirect co-culture to induce the differentiation of hUCMSCs. METHODS: hUCMSCs were isolated in vitro and then expanded in culture medium. After the treatment of mitomycin C, the cells were seeded on porous membranes of 6-well-dish Transwell inserts with different pore sizes of 0.4, 3.0 and 8.0 µm respectively. After culturing for 7 days, the cells were observed and counted on the bottom of each porous membrane. Then the calculation of migration ratio was performed. The growth and migration of hUCMSCs on porous membranes were also examined under scanning electron microscope (SEM). RESULTS: The migration ratios of hUCMSCs on membranes of 0.4, 3.0 and 8.0 µm pore sizes were 0, 1.8% and 8.0% respectively. The migration ratio of cells on 0.4 µm pore size membrane was statistically different from that of the other two pore size groups (P < 0.01). Under SEM, a small portion of cells were growing on the bottoms of membranes and moving through the pores. But there was no cell movement through 0.4 µm pore size membrane. CONCLUSIONS: hUCMSCs can migrate through the polycarbonate membranes of 3.0 µm and 8.0 µm pore sizes but not through the 0.4 µm one. Thus both sides of polycarbonate membrane of 0.4 µm pore size may be used for close indirect co-culture to induce the differentiation of hUCMSCs.

[Early changes in serum neutrophil elastase in rats with burn, blast injury or combined burn-blast injury and its significance].

OBJECTIVE: To explore the early changes in serum neutrophil elastase (NE) in rats with burn, blast injury or combined burn-blast injury and its significance. METHODS: A total of 176 male Sprague Dawley rats were randomly divided into four groups: control (C), burn (BU), blast injury (BL) and burn-blast combined injury (BB). Rats in C group were not injured. Animals in BU group were subjected to 25% TBSA full-thickness burn on back with 94 degrees C water for 12 seconds; Animals in BL group were inflicted with moderate blast injury with 5 g 8701 compressed dynamite stick as the explosion source 75 cm away while left chest facing the explosive source; Rats in BB group were burned immediately after the blast injury similarly as in BL group. During the first 24 h post-injury, animals in BU and BB groups received intraperitoneal injection of sodium lactate Ringer's solution at a dose of 50 ml x kg(-1) x 12 h(-1). Protein concentration in bronchoalveolar lavage fluid (BALF), water content of lung tissue and NE content in serum were determined at 0 h (C group), 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 7 d post-injury. RESULTS: Protein concentration in BALF, water content of lung tissue and NE content in serum in SD rats of the injured groups were significantly higher than those in C group (P < 0.01 or P < 0.05), peaked within 2 d post-injury, especially at 2 d post-injury (NE content in serum: BU group, 319. 85 +/- 19.50 ng/ml; BL group, 467.43 +/- 31.64 ng/ml; BB group, 626.00 +/- 26.38 ng/ml vs. C group, 78.53 +/- 25.10 ng/ml). Overall, protein concentration in BALF, water content of lung tissue and NE content in serum in BB group were significantly higher than BU and BL groups (P < 0.01 or P < 0.05). Correlation analysis showed that within 3 d postinjury, a significant positive correlation was found between the protein concentration in BALF, water content of lung tissue and NE content in serum (r = 0.7910, 0.8078, P < 0.05) in BU group. NE content in serum and protein concentration in BALF were significantly positively correlated in BB group (r = 0.8672, P < 0.05). CONCLUSION: NE may play an important role in early lung injury of burn or blast injury, especially in combined burn-blast injury.

Isolation, culture, and verification of human sweat gland epithelial cells.

Human sweat gland epithelial cells (SGECs) have been isolated and grown in vitro, However, slow proliferation makes the culture of these cells extremely difficult. The present study was carried out to explore the modified culture medium for SGECs in vitro. Full-thickness skin samples were minced (1 mm(3)) and digested overnight with type II collagenase. The gland coils were removed under an inverted phase-contrast microscope. An adherent culture method was used to isolate and culture SGECs. Staining with hematoxylin and eosin was performed, followed by observation of the morphologic features of these cells. Immunofluorescence staining with antibodies to cytokeratins CK7, CK18, and CK19 and carcinoembryonic antigen (CEA) was performed to verify the presence of SGECs. Growth curves by MTT were created for cells grown in serum-free keratinocyte medium and in modified keratinocyte medium containing 2.5% fetal bovine serum (FBS). One week after culturing, the cells grew well and were polygonal or irregular in shape by inverted phase contrast microscopy. Cell fusion, with a characteristic paving-stone arrangement, reached 100% after approximately 3 weeks in culture. Immunofluorescence staining indicated expression of CK7, CK18, CK19, and CEA. Compared with SGECs grown in serum-free keratinocyte medium, the proliferation of SGECs grown in modified culture medium with low concentration of FBS at days 6, 9, and 12 was significantly accelerated (p < 0.05). This study suggests that keratinocyte medium supplemented with 2.5% FBS is effective and suitable for the culture of SGECs.

[Research progress of full-thickness tissue engineered skin].

OBJECTIVE: To review the latest research progress of full-thickness tissue engineered skin (FTTES), to thoroughly understand its current state of research and application so as to lay a solid foundation for developing new type FTTES and improving the quality of skin substitutes. METHODS: Domestica and international literature concerning FTTES in recent years was extensively reviewed and comprehensively analyzed. RESULTS: Some progress of FTTES had made in seed cells, scaffold materials and construction, and some therapeutic efficacy had also been achieved in clinical application. But FTTES grafting successful rate was lower, and it had no complete skin structure and had not reached the requirements of clinical application. CONCLUSION: FTTES is an ideal skin substitute and has great development prospects. However, in seed cells, scaffold materials, construction and applications of FTTES, further studied is still needed.

[Clinical application and pathological observation of acellular allogeneic dermal matrix in repairing unstable burn scar].

OBJECTIVE: To evaluate the clinical effect and the pathological characteristics of acellular allogeneic dermal matrix in repairing unstable burn scar. METHODS: From January 2007 to June 2008, 19 cases of unstable burn scars (24 parts) were treated, including 16 males (20 parts) and 3 females (4 parts) with a median age of 27 years (range, 3-58 years). The injury was caused by flame (14 cases, 18 parts), electricity (4 cases, 5 parts), and hot water (1 case, 1 part). The unstable burn scars located on hands (8 cases), forearms (2 cases), thighs (3 cases), legs (2 cases), feet (2 cases), chest (1 case), and abdomen (1 case). Scar formed for 3 months to 1 year. The area of defect varied from 7 cm x 5 cm to 22 cm x 15 cm after scar removal. Defects were covered with acellular allogeneic dermal matrix and autogenous split-thickness skin graft. At 6-18 months after operation, the pathological observations of the epidermis, the basal membrane, and structural components of the dermis were done. RESULTS: All wounds healed by first intention. Scar ulcer disappeared completely in 18 cases and the composite skin grafts all survived. Some blisters occurred in 1 case and were cured after dressing changing. All patients were followed up 10 months to 2 years (18 months on average). The grafted-skin was excellent in the appearance, texture, and elasticity. The function recovered well. Only superficial scar was observed at skin donor sites. Pathological observation showed that the epidermis and the basal membrane of the skin grafts were similar to that of normal skin, and no significant difference was found in newly capillaries between them. Collagen fibers arranged regularly, and there were few inflammatory cells in the matrix. CONCLUSION: Acellular allogeneic dermal matrix with autogenous split-thickness skin graft may effectively repair the wound after removing the unstable burn scar, and its structure is similar to that of normal skin.

[Preparation of selectively decellular xenoskin and its biocompatibility].

OBJECTIVE: To prepare and study the biocompatibility of selectively decellular xenoskin which has the character of the lower antigen, continuous epidermis, and the dermal matrix without any cellular components. METHODS: The porcine skin was treated with glutaraldehyde solution, trypsin, and detergent solution TritonX-100 to prepare the selectively decellular xenoskin. The cytotoxicity was tested according to GB/T16886.5-2003 biological evaluation of medical devices for in vitro cytotoxicity, and the levels of cytotoxicity were evaluated with the United States Pharmacopeia. Subdermal implantation was tested according to GB/T16886.6-1997 biological evaluation of medical devices for local effects after implantation. Seventy-two mature Wistar rats were randomly assigned to groups A, B, and C (n = 24). Three kinds of materials were implanted into subcutaneous of rats back. Selectively decellular xenoskin was transplanted into group A, fresh porcine skin was transplanted into group B, and allogeneic skin was transplanted into group C. The samples were collected to make the observation of gross and histology after 1, 2, 4, 8, 12, and 16 weeks. RESULTS: The cytotoxicity was proved to be first grade by biocompatibility test. The gross and histological observation of subdermal implantation: after implantation, the most severe inflammatory reactions were seen in group B which dispersion was very slow. Inflammatory reactions in groups A and C alleviated gradually. In groups A and C, there was an increased collagen fiber density and angiogenesis at late stage; the transplanted skin was gradually degraded and absorbed. In group B, no obvious degradation and absorption were observed. CONCLUSION: Selectively decellular xenoskin, prepared with glutaraldehyde solution, trypsin, and detergent solution, possesses characteristics of integral skin structure and excellent biocompatibility, so it can be used as a new type substitute to repair the burn wound.

[Autologous free fat particle grafting combined with bFGF to repair facial depression].

OBJECTIVE: To evaluate the effect of autologous free fat particle grafting combined with-bFGF to repair facial depression. METHODS: From April 2004 to May 2006, 41 patients with facial depression were randomized into two groups (groups A and B). In group A, 12 cases were admitted from April 2004 to December 2004. There were 5 males and 7 females, aging 16-49 years (mean, 31 years). The pathological causes were congenital facial depression in 2 patients, hemifacial atrophy in 2, traumatic cicatrix in 5 and benign tumor removal in 3. The course of disease was 2-19 years. The concave regions were low (0.52 +/- 0.13) cm compared to surrounding normal skin, concave area (16.0 +/- 5.3) cm2. In group B, 29 cases were admitted from January 2005 to May 2006. There were 14 males and 15 females, aging 18-52 years (mean, 37 years). The pathological causes were: congenital facial depression in 3 patients, hemifacial atrophy in 4, traumatic cicatrix in 15 and benign tumor removal in 7. The course of disease was 2-20 years. The concave regions were low (0.58 +/- 0.15) cm compared to surrounding normal skin, concave area (18.0 +/- 6.2) cm2. Cases in group A were treated with pure autologous free fat particle injection; cases in group B were treated with autologous free fat particle injection combined with bFGF (4,200 IU/10 mL). The clinical outcome were comparatively analyzed between two groups after operation. RESULTS: The follow-up time was 6 to 24 months (mean, 12.5 months) in group A and 6 to 24 months (mean, 13 months) in group B. In group A, 6 patients achieved satisfactory clinical effect after one injection of fat particle, the satisfactory rate of one therapy being 50%; other 6 cases were required reinjection of fat particle 6-12 months postoperatively, of which two-time injections in 3 cases, three-time injections in 3 cases. In group B, 24 patients achieved satisfactory clinical effect after one injection of fat particle, the satisfactory rate of one therapy being 82.8%; only 5 cases were required reinjection one year postoperatively. There was statistically significant difference in the satisfactory rate of one injection between two groups (P < 0.05). CONCLUSION: Autologous free fat particle grafting combined with bFGF to treat facial depression can acquire satisfactory clinical effect, which is a safe and effective method.