Phenotypic and Cellular Characteristics of a Stromal Vascular Fraction/Extracellular Matrix Gel Prepared Using Mechanical Shear Force on Human Fat.

The retention of fat-derived grafts remains a challenge for regenerative medicine. Fat aspirates from patients undergoing liposuction were prepared into standard Coleman fat grafts or further isolated using mechanical shear force to prepare a stromal vascular fraction (SVF)/extracellular matrix (ECM) gel. The retention rate of the SVF/ECM gel was significantly higher than that of the Coleman fat at 3, 14, 28, and 60 days following transplantation on the backs of nude mice. The viscosity of the fat was directly proportional to the shearing force. Although the mechanical isolation did not affect the total number of cells, it significantly decreased the number of living cells. Flow cytometry showed a greater number of mesenchymal stem cells, supra-adventitial (SA)-adipose stromal cells (ASCs), and adipose-derived stem cells but a lower number of endothelial progenitor cells in the SVF/ECM gel than in the Coleman fat. Thus, mechanical isolation of fat can increase the pluripotency of adipocytes, which can improve graft retention in cell therapy.

Tear trough deformity: different types of anatomy and treatment options.

AIM: To explore the efficacy of tear trough deformity treatment with the use of hyaluronic acid gel or autologous fat for soft tissue augmentation and fat repositioning via arcus marginalis release. MATERIAL AND METHODS: Seventy-eight patients with the tear trough were divided into three groups. Class I has tear trough without bulging orbital fat or excess of the lower eyelid skin. Class II is associated with mild to moderate orbital fat bulging, without excess of the lower eyelid skin. Class III is associated with severe orbital fat bulging and excess of the lower eyelid skin. Class I or II was treated using hyaluronic acid gel or autologous fat injections. Class III was treated with fat repositioning via arcus marginalis release. The patients with a deep nasojugal groove of class III were treated with injecting autologous fat into the tear trough during fat repositioning lower blepharoplasty as a way of supplementing the volume added by the repositioned fat. RESULTS: Seventy-eight patients with tear trough deformity were confirmed from photographs taken before and after surgery. There were some complications, but all had complete resolution. CONCLUSIONS: Patients with mild to moderate peri-orbital volume loss without severe orbital fat bulging may be good candidates for hyaluronic acid filler or fat grafting alone. However, patients with more pronounced deformities, severe orbital fat bulging and excess of the lower eyelid skin are often better served by fat repositioning via arcus marginalis release and fat grafting.

Congenital arhinia: A rare case.

PATIENT: Male, 4 months FINAL DIAGNOSIS: Congenital arhynia Symptoms: Absence of the nose Medication: - Clinical Procedure: - Specialty: Pediatrics and Noenatology • Genetics. OBJECTIVE: Congenital defects. BACKGROUND: Congenital nasal absence (arhinia) is an extremely rare malformation. Arhinia causes severe airway obstruction and poor feeding in the affected neonate. There is an association with other facial anomalies, especially defects of the eyes, ears, palate, and midline defects. CASE REPORT: A full-term boy was born via an uncomplicated vaginal delivery. The mother was 40 years old and had a normal pregnancy. The mother had 4 previous uncomplicated pregnancies. There was no history of drug use during pregnancy. CONCLUSIONS: Congenital arhinia is a rare defect of embryogenesis, often associated with other anomalies that significantly influence the immediate and long-term outcomes of the neonate. It is a potentially life-threatening condition and requires the presence of a highly skilled neonatal resuscitation team at the time of delivery. Parental counseling is vital and a multidisciplinary team approach is required to optimize neonatal outcome.

Compressed collagen gel as the scaffold for skin engineering.

Collagen gel scaffolds can potentially be utilized as cell seeded systems for skin tissue engineering. However, its dramatic contraction after being mixed with cells and its mechanical weakness are the drawbacks for its application to skin engineering. In this study, a compressed collagen gel scaffold was fabricated through the rapid expulsion of liquid from reconstituted gels by the application of 'plastic compression'(PC) technique. Both compressed and uncompressed gels were characterized with their gel contraction rate, morphology, the viability of seeded cells, their mechanical properties and the feasibility as a scaffold for constructing tissue-engineered skin. The results showed that the compression could significantly reduce the contraction of the collagen gel and improve its mechanical property. In addition, seeded dermal fibroblasts survived well in the compressed gel and seeded epidermal cells gradually developed into a stratified epidermal layer, and thus formed tissue engineered skin. This study reveals the potential of using compressed collagen gel as a scaffold for skin engineering.

Skin epithelial cells in mice from umbilical cord blood mesenchymal stem cells.

UNLABELLED: The purpose of this study was investigation of the potential to isolate mesenchymal stem cells (MSCs) from human umbilical cord blood (UCB) and differentiate them into epithelial cells in mouse skin tissues. Mononuclear cells (MNCs) from UCB (UCB-MNCs) were isolated and induced to MSCs in culture. UCB-MSCs were transfected with pEGFP and labeled with PKH26 dye. eGFP-transfected and PKH26-labeled UCB-derived MSCs were purified by flow cytometry to gate purified eGFP(+) PKH26(+) cells and transplanted at a single clone level into injured nude Balb/C mice by tail vein injection. The phenotype of cultured UCB-MSCs, the expression of human cytokeratins and the expression of eGFP(+) PKH26(+) cells in mouse skin tissues were examined by flow cytometry. Human HLA-1 antigen and cytokeratin 10 (CK10) were detected by direct immunofluorescence on mouse skin tissue sections and flow cytometry. Sry gene (sex-determining region of Y chromosome) was detected by PCR reaction. The results showed that MSCs were isolated from UCB and had heterogeneous morphology and growth potential. Moreover, UCB-derived MSCs localized into mouse skin tissues and differentiated into skin epithelial cells confirmed by in vivo cell tracking and human antigen detection. At two weeks after transplant, a number of eGFP(+) PKH26(+) cells were detected in recipient mouse skin tissues. The detection of sry gene and HLA-1 antigen further confirmed that the human UCB-derived cells were present in recipient mouse skin tissues. Human cells localizing to mouse skin and differentiating into skin epithelial cells were demonstrated by cytokeratins (CK) 8 and 10 expression during flow cytometry, and CK10 expression on injured skin tissue section by direct immunofluorescence. CONCLUSION: UCB-derived MSCs localized to injured skin in vivo and differentiated into epithelial phenotypes. The results demonstrate that UCB-derived MSCs contribute to skin tissue regeneration in vivo and may be an ideal cell source for therapy of skin epithelial tissue injury, including burns.

[Construction of a tissue engineering skin with epidermal stem cells].

OBJECTIVE: To constitute a composite skin substitute that can proliferate well with epidermal stem cells and fibroblasts on collagen sponge. METHODS: Epidermal stem cells were selected by rapid attachment to collagen IV for 10-15 min and cultured on 3T3 feeder layers. Collagen was extracted from rat tail. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde. Fibroblasts were inoculated on collagen sponge and cultured for 4 days prior to inoculation of epidermal stem cells to construct composite skin substitute. The composite skin substitute were examined by means of histology, immunohistochemistry and electron microcopy, the histologic appearance was similar to that of normal epidermis. RESULTS: The epidermal stem cells formed large colonies at 7-8 days, expressed K19 antigen. The percentages of cells at G0/G1 phase of cell cycle and the percentage of alpha6 briCD71dim cells in ESC groups were higher than those in the control group. The skin substitute had epidermis and dermis, the histologic appearance was similar to that of normal skin. The artificial skin expressed keratin antigen by immunocytochemical methods. CONCLUSIONS: Epidermal stem cells proliferated well and differentiated properly on this artificial skin dermis which contained fibroblasts. It seemed that the composite skin to be a good equivalent.

An experimental study on the repair of full skin loss of nude mice with composite graft of epidermal stem cells.

This study is to constitute a composite skin substitute with epidermal stem cells (ESCs) and fibroblasts on collagen sponge. ESCs were selected by rapid attachment to collagen IV for 10 min. Collagen was extracted from rat's tail. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde. Fibroblasts were inoculated on collagen sponge and cultured for 1 week prior to inoculation of ESCs. Having cultured for 2 weeks in submerged culture, the bioengineered tissue was raised to the air-liquid interface and cultured for 2 weeks. The artificial skin was then grafted onto full skin loss wounds of nude mice. Collagen sponge membrane lacking cell inoculation and an artificial skin with epidermal cells (ECs) and fibroblasts were used as controls. The wounds were observed daily. Tissue samples were harvested and examined by means of histology, immunohistochemistry and electron microscopy. The wounds in the test group healed at a significantly faster rate than controls, with good skin appearance and minimal scar formation. The control group showed delayed wound healing and intensive wound contraction as compared to the test group. Thus the skin substitute with ESCs seemed to be a good equivalent.

[Separation and culture of human epidermal stem cells in vitro].

OBJECTIVE: To investigate the culture method for epidermal stem cells in vitro. METHODS: The epidermis was separated from the dermis, and shaken for 10 min in 0.05% trypsin at 37 C to dissociate into single cells. Epidermal stem cells were selected by rapid attachment to collagen IV for 10-15 min and cultured on collagen IV or 3T3 feeder layer. All the cells were grown in DMEM without calcium, supplemented with 10% chelexed fetal bovine serum, 10 microg/L epidermal growth factor, 0.05 mmol/L CaCl2 and 0. 8 mg/L hydrocortisone. Cultures were observed for colony formation under a phase contrast microscope. The phenotypes of epidermal stem cells were detected by flow cytometry and immunocytochemistry staining. RESULTS: The cells selected by rapid adherence to collagen IV formed large colonies at 7- 8 days, expressed K19 antigen. The percentages of cells at the G0 and G1 phases of the cell cycle and the percentage of alpha 6 briCD71 dim cells in the experimental groups were higher than those in the control group. It indicated that there was a significant difference between the experimental groups and the control groups(P<0. 05). CONCLUSION: The human epidermal stem cells can be selected by rapid attachment to collagen IV, and they can be expanded in culture if the appropriate conditions are maintained.