Decellularized Human Adipose Tissue as an Alternative Graft Material for Bone Regeneration.

BACKGROUND: Tissue engineering approaches to treat damaged bone include various tissue transplants such as autologous, allogeneic, and xenografts. Artificial materials have been widely introduced to meet the demand for graft materials, but insufficiency in supply is still not resolved. In this study, human adipose tissue, easily obtained from the human body, was harvested, and the tissue was decellularized to fabricate a decellularized human adipose tissue matrix (DM) as an alternative graft material. METHODS: Human adipose tissue was obtained via liposuction. The obtained fresh adipose tissue sample was cut into pieces then put into decellularization solution (1% antibiotic-antimycotic solution and 1% phenylmethanesulphonyl fluoride). Lipids were further removed via treatment in isopropanol. The sample was then subjected to another enzymatic digestion and lipid removal processes. The obtained decellularized adipose tissue matrix was lyophilized to form a graft material in disc shape. RESULTS: Decellularization was confirmed by nuclear staining methods and detection of RNA and DNA via PCR. Bone morphogenetic protein 2 (BMP2)-loaded DM showed the ability to form new bone tissue when implanted in subcutaneous tissue. In recovery of a mouse calvarial defect model, BMP2-loaded DM exhibited similar levels of bone tissue regeneration efficiency compared with a well-defined commercial product, BMP2-loaded CollaCote®. CONCLUSION: The DM developed in this study is expected to address the problem of insufficient supply of graft materials and contribute to the treatment of bone defects of critical size as an alternative bone graft material with preserved extracellular matrix components.

Early Treatment Effects of Nonablative Fractional Lasers (NAFL) on Hypertrophic Scars in an Animal Model.

BACKGROUND AND OBJECTIVES: Recently, there have been several attempts to apply the laser therapy to hypertrophic scars (HTS). In particular, the fractional laser is in the spotlight for its usefulness in rapid wound healing and dermal remodeling. However, most previous studies have focused on the ablative fractional laser (AFL), and there are no studies on the mechanism of the nonablative fractional laser (NAFL) effect in HTS treatment. In this study, we aimed to evaluate the changes in histology and molecular chemistry to provide scientific evidence for the early treatment of HTS with NAFL. STUDY DESIGN/MATERIALS AND METHODS: A total of 40 hypertrophic burn scars were made on the abdomens of two female pigs. After epithelialization, the HTS were randomly subdivided into four groups-control, AFL, NAFL (low energy), and NAFL (high energy). Laser treatment was initiated 1 week after the crust fell and the epithelium became covered, and it was repeated for six sessions over an interval of 2 weeks. Five excisional biopsies were obtained for histologic analysis and biomarker assessment. RESULTS: Histologically, dermal remodeling with thin coil-shaped collagen fibers was observed in the NAFL groups. It also showed a significant increase of matrix metalloproteinase-2 (MMP-2) and Decorin at 16 weeks in an enzyme-linked immunosorbent assay. The reverse-transcription polymerase chain reaction analysis showed a tendency that high-pulse energy of NAFL led to higher messenger RNA expression than did the low-energy group. CONCLUSION: The NAFL-treated groups showed characteristic collagen re-arrangement and a significant increase in MMP-2 and Decorin. These molecular changes suggest that MMP-2 and Decorin play a significant role in dermal remodeling. Early NAFL treatment for HTS could be supported with both histological and molecular evidence. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.

The Relationship of a Combination of Human Adipose Tissue-Derived Stem Cells and Frozen Fat with the Survival Rate of Transplanted Fat.

BACKGROUND: The survival rate of grafted fat is difficult to predict, and repeated procedures are frequently required. In this study, the effects of the freezing period of harvested adipose tissue and the addition of human adipose tissue-derived stem cells (ASCs) on the process of fat absorption were studied. METHODS: Adipose tissue was obtained from patients who underwent a lipoaspirated fat graft. The fat tissue was cryopreserved at -20℃ in a domestic refrigerator. A total of 40 nude mice were used. The mice in the experimental group received three different subcutaneous injections in the back: an injection of fresh fat and ASCs, an injection of fat that had been frozen for one month and ASCs, and an injection of fat that had been frozen for two months and ASCs. The control mice received fat grafts without ASCs. The mice were sacrificed at four or eight weeks after the procedure, and the grafted fat tissues were harvested. The extracted fat was evaluated using photographic analysis, volume measurements, and histological examination. RESULTS: In the control group, the fat resorption rates four weeks after transplantation in the grafts of fresh fat, fat that had been frozen for one month, and fat that had been frozen for two months were 21.14%, 22.46%, and 42.56%, respectively. In the experimental group, the corresponding resorption rates were 6.68%, 13.0%, and 33.9%, respectively. CONCLUSIONS: ASCs can increase the fat graft survival rate. The use of ASCs in fat grafting can reduce the need for repeated fat grafts and provide good long term results.

Effects of platelet-rich plasma, adipose-derived stem cells, and stromal vascular fraction on the survival of human transplanted adipose tissue.

Traditional adipose tissue transplantation has unpredictable viability and poor absorption rates. Recent studies have reported that treatment with platelet-rich plasma (PRP), adipose-derived stem cells (ASCs), and stromal vascular fraction (SVF) are related to increased survival of grafted adipose tissue. This study was the first simultaneous comparison of graft survival in combination with PRP, ASCs, and SVF. Adipose tissues were mixed with each other, injected subcutaneously into the back of nude mice, and evaluated at 4, 8, and 12 weeks. Human adipocytes were grossly maintained in the ASCs and SVF mixtures. Survival of the adipose tissues with PRP was observed at 4 weeks and with SVF at 8 and 12 weeks. At 12 weeks, volume reduction in the ASCs and SVF mixtures were 36.9% and 32.1%, respectively, which were significantly different from that of the control group without adjuvant treatment, 51.0%. Neovascular structures were rarely observed in any of the groups. Our results suggest that the technique of adding ASCs or SVF to transplanted adipose tissue might be more effective than the conventional grafting method. An autologous adipose tissue graft in combination with ASCs or SVF may potentially contribute to stabilization of engraftment.

In vivo hair growth-promoting effect of rice bran extract prepared by supercritical carbon dioxide fluid.

The potential hair growth-promoting activity of rice bran supercritical CO2 extract (RB-SCE) and major components of RB-SCE, linoleic acid, policosanol, γ-oryzanol, and γ-tocotrienol, were evaluated with the histological morphology and mRNA expression levels of cell growth factors using real-time reverse transcriptase-polymerase chain reaction (PCR) in C57BL/6 mice. RB-SCE showed hair growth-promoting potential to a similar extent as 3% minoxidil, showing that the hair follicles were induced to be in the anagen stage. The numbers of the hair follicles were significantly increased. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and keratinocyte growth factor (KGF) were also significantly increased and that of transforming growth factor-ß (TGF-ß) decreased in RB-SCE-treated groups. Among the major components of RB-SCE, linoleic acid and γ-oryzanol induced the formation of hair follicles according to examination of histological morphology and mRNA expression levels of cell growth factors. In conclusion, our results demonstrate that RB-SCE, particularly linoleic acid and γ-oryzanol, promotes hair growth and suggests RB-SCE can be applied as hair loss treatment.

Effects of Lycopersicon esculentum extract on hair growth and alopecia prevention.

To evaluate the potential hair growth-promoting activity and the expression of cell growth factors of Lycopersicon esculentum extracts, each 3% (w/w) of ethyl acetate extract (EAE), and supercritical CO2 extract (SCE) of L. esculentum and isolated lycopene Tween 80 solution (LTS) and test hair tonic (THT) containing LTS were applied on the dorsal skin of C57BL/6 mice, once a day for 4 weeks. At week 4, LTS and THT exhibited hair growth-promoting potential similar to that of 3% minoxidil as a positive control (PC). Further, in the LTS group, a significant increase of mRNA expression of vascular endothelial growth factor (VEGF), keratinocyte growth factor, and insulin-like growth factor-1 (IGF-1) was observed than PC, as well as the negative control (NC). In the THT group, increases in IGF-1 and decrease in VEGF and transforming growth factor-ß expression were significant over the NC. In a histological examination in the THT group, the induction of anagen stage of hair follicles was faster than that of NC. In the Draize skin irritation study for THT, no observable edema or erythema was observed on all four sectors in the back skin after exposure for 24 or 72 h for any rabbit. Therefore, this study provides reasonable evidence that L. esculentum extracts promote hair growth and suggests that applications could be found in hair loss treatments without skin irritation at moderate doses.

Transplantation of adipose derived stem cells for peripheral nerve regeneration in sciatic nerve defects of the rat.

Tissue engineering approaches for promoting the repair of peripheral nerve injuries have focused on cell-based therapies involving Adipose-derived stem cells (ASCs). The authors evaluated the effects of undifferentiated ASCs and of neurally differentiated ASCs on the regenerating abilities of peripheral nerves. We hope that this would demonstrate the feasibility of using adipose derived stem cells for peripheral nerve regeneration and provide clues regarding the use of adipose- derived stem cells. ASCs were isolated and cultured. Then the cells were cultured with neuronal induction agents for neural differentiation. ASCs and neurally differentiated ASCs were transplanted into sciatic nerve defects. After 12 weeks, the number and diameter of the myelinated fibers were measured and nerve conduction study was done. The extent of regeneration of myelinated fibers in the neurally differentiated ASCs transplanted group was greater than that in the ASCs transplanted group or the control group. However, thickness of myelin sheath and diameter of nerve fibers in the ASCs transplanted group were greater than those in the neutrally differentiated ASCs transplanted group or the control group. Nerve conduction study showed good recovery in the neurally differentiated ASCs transplanted groups. Muscles can atrophy and contract if denervation has started. It would be difficult to recover muscle function even if the nerve was reinnervated. Therefore, although neurally differentiated ASCs were found to have a greater functional effect than non-differentiated ASCs, time constraint is important when considering a method of ASCs transplantation.

Enhancement of bone regeneration using osteogenic-induced adipose-derived stem cells combined with demineralized bone matrix in a rat critically-sized calvarial defect model.

INTRODUCTION: Human adipose tissue contains pluripotent stem cells that are similar to bone marrow-derived stem cells. The present study examined whether osteogenic induced adipose-derived stem cells (ASCs) could enhance the osteogenic capacity of demineralized bone matrix and accelerate bone formation in a rat critically-sized calvarial defect model. MATERIALS AND METHODS: Forty Sprague-Dawley rats were divided randomly into four groups containing 10 rats per each group (Control, 0.05 cc fibrin glue (25 mg/ml) and 0.05 cc thrombin (130 U/ml); DBX, control + 0.2 g DBX�; ASC, DBX + 1 x 105 ASCs/g; iASC, DBX + 1 x 105 osteogenic-induced ASCs/g). After osteogenic differentiation of ASCs, alkaline phosphatase and von Kossa staining were performed each week to determine the extent of differentiation and mineralization. An 8-mm critical size circular defect was made in the calvarial bone of each rat. The specimens were harvested 8 weeks after implantation, and radiographic and histological evaluations were carried out. New bone formation was quantified by radiodensitometric analysis of the calvarial sections. Statistical analysis was accomplished using a Mann-Whitney test and Kruskal-Wallis test at a significance level of P < 0.05. RESULTS: Alkaline phosphatase and von Kossa staining showed that the osteogenic-induced ASCs yielded higher osteogenic differentiation at 3 weeks. The calvarial defect was filled more in the iASC group compared to the other groups, as demonstrated by the gross appearance of the specimen and radiologic evaluation. The mean radiodensity of the control, DBX, ASC, and iASC group was 16.78%, 39.94%, 25.58%, and 51.31%, respectively, and these were significantly different (P=0.034). Histomorphological evaluation confirmed that new bone formation was accelerated and enhanced by the osteogenic-induced ASCs. CONCLUSIONS: ASCs produced greater osteogenic differentiation at 3 weeks. Osteogenic regeneration was accelerated and enhanced in vivo with the osteogenic-induced ASCs, compared to undifferentiated ASCs. Osteogenic-induced ASCs are an excellent and promising candidate for regenerative medicine and tissue engineering application.

Vascular complications of hyaluronic acid fillers and the role of hyaluronidase in management.

Skin necrosis following the inadvertent arterial injection of hyaluronic acid (HA) is a serious complication. It is not clear whether or not subcutaneous injections of hyaluronidase decrease skin necrosis in HA-induced vascular complications. We had four cases of HA-induced vascular complications, two of which were treated with hyaluronidase the next day. All of the patients had skin necrosis and scarring. We performed an animal study with rabbit ears in which HA filler was injected into the auricular arteries of both ears. Five rabbits each received a subcutaneous injection of 750 IU of hyaluronidase 4 and 24 h after the filler injection. The hyaluronidase-treated ears in the 4-h intervention group had significantly smaller necrotic areas (p<0.05), while the 24-h intervention group had no differences in the area of necrosis. Hyaluronidase reduced the vascular complications of HA fillers when used early, but there was no benefit to hyaluronidase injection after 24 h.

Morphine attenuates endothelial cell adhesion molecules induced by the supernatant of LPS-stimulated colon cancer cells.

A large reservoir of bacterial lipopolysaccharide (LPS) is available in the colon and this could promote colon cancer metastasis by enhancing tumor cell adhesion, intravasation, and extravasation. Furthermore, adhesion molecules like ICAM-1, VCAM-1, and E-selectin play important roles in the adhesion of tumor cells to endothelium. This study was designed to determine whether morphine can attenuate the expressions of adhesion molecules up-regulated by the supernatant of LPS-stimulated HCT 116 colon cancer cells (LPS-Sup). In this study, we divided to three groups by cell-growth medium of human umbilical vascular endothelial cells (HUVECs): the control group was incubated in growth factor-free endothelial medium, the Sup group was incubated in the supernatant of HCT 116 cells (Sup), and the LPS-Sup group was incubated in LPS-Sup. To observe effect of morphine to the adhesion molecules expressions in the LPS-Sup group, we co-treated morphine with LPS or added it to LPS-Sup. Adhesion molecule expressions on HUVECs in all three groups were measured during incubation period. Consquentially, ICAM-1, VCAM-1, and E-selectin expressions on HUVECs were significantly lower when morphine was co-treated with LPS than not co-treated. Thus, we suggest that morphine affects the expressions of adhesion molecules primarily by attenuating LPS stimuli on tumor cells.

In vivo evaluation of mixtures of uncultured freshly isolated adipose-derived stem cells and demineralized bone matrix for bone regeneration in a rat critically sized calvarial defect model.

Although many studies have suggested that human adipose tissue contains pluripotent stem cells, a few reports are available on stromal vascular fraction (SVF). In the present study, we evaluated the bone formation capacities of SVF. We implanted uncultured freshly isolated adipose-derived stem cells combined with demineralized bone matrix (DBM) to induce bone regeneration in a critically sized rat calvarial defect model. We used DBM (DBX(®)) and/or poly(70L-lactide-co-30DL-lactide) copolymer PLA as a scaffold. Fifty white rats were randomized to 5 different groups (n=10): (1) control, (2) DBM, (3) DBM + SVF, (4) DBM + PLA, and (5) DBM + PLA + SVF groups. After acquiring SVF, an 8-mm critically sized calvarial defect was made in each rat. Specimens were harvested at 8 weeks postimplantation and evaluated radiographically and histologically. New bone formation was qualified by hematoxylin and eosin staining and anti-osteocalcin antibody (OC4-30) immunostaining of calvarial sections. Amounts of mineralization were determined by radiodensitometric analysis. In gross appearance, the DBM + SVF and DBM + PLA + SVF groups showed more abundant bone formation than the other groups. Radiodensitometric evaluations revealed that significant intergroup differences were observed according to the Kruskal-Wallis (rank) test (P=0.030<0.05). The 5 groups show different amounts of filling of bone defects (control: 13.48%; DBM: 39.94%; DBM + SVF: 57.69%; DBM + PLA: 24.86%; DBM + PLA + SVF: 42.75%). Histological evaluation revealed that there was abundant new bone formation in the DBM + SVF and DBM + PLA + SVF groups. It was found that undifferentiated adipose-derived stem cells in the form of SVF induced new bone formation in rat calvarial defects. Accordingly, SVF offers a practical, promising candidate for regenerative tissue engineering or cell-based therapy.

Vascular endothelial growth factor (VEGF) and soluble VEGF receptor FLT-1 in diabetic nephropathy.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors have been implicated in the pathogenesis of diabetic nephropathy. The objective of this study was to determine whether alterations of the plasma and urinary VEGF and sFLT-1 levels were related to the stages and risk factors of diabetic nephropathy. In addition, we also examined the regulation of the VEGF/sFLT-1 expression by various stimuli in cultured human proximal tubule cells (HPTC). METHODS: A total of 107 type 2 diabetic patients and 47 healthy control subjects were studied. The expression and protein levels of VEGF and sFLT-1 were measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The urinary VEGF and sFLT-1 excretions were significantly increased in the microalbuminuric and proteinuric diabetic patients. The urinary VEGF levels were positively correlated with the urinary albumin to creatinine ratio (ACR), urinary sFLT-1 levels, and negatively correlated with creatinine clearance. The urinary sFLT-1 levels also showed a positive relationship with the urinary ACR. In cultured HPTC, high glucose stimuli rapidly up-regulated VEGF synthesis without having any effect on sFLT-1 synthesis. Interestingly, angiotensin II (Ang II) induced a dose-dependent increase in the synthesis of both VEGF and sFLT-1, which was significantly blocked by losartan. CONCLUSION: The urinary excretion of VEGF and sFLT-1 increased at a relatively early stage in diabetic nephropathy associated with urinary albumin excretion. A marked increase in both VEGF/sFLT-1 synthesis in response to Ang II was observed in HPTC, which was different from the response to glucose stimuli. These findings may imply that VEGF and sFLT-1 can actively take part in the pathogenesis of diabetic nephropathy.