Cryopreservation of Fat Tissue and Application in Autologous Fat Graft: In Vitro and In Vivo Study

Background

Absorption of the autologous fat graft results in repeated harvesting procedures. The cost and complications increase with repeated procedures, but cryopreservation is one way to solve the problem. The aim of this study was to find an optimal temperature at which to store fat tissue with or without cryoprotective agents for long-term use.

Methods

Fat tissues harvested by liposuction were stored in normal saline, frozen in the freezer following the preset program, and cryopreserved at ?20, ?80, and ?196°C. The other group of fat tissues was stored in hydroxyethyl starch using the same frozen procedure. Two and 7 days after cryopreservation, viability tests were conducted. The fat tissues were injected into nude mice 2 and 4 weeks after cryopreservation. Three months later the fat grafts were harvested for histologic examination.

Results

No significant differences in cell viability were found in either in vitro or in vivo experiments for the three preserving temperatures. The cryoprotective agent HES did not influence cell viability.

Conclusion

There were no differences in cell viability among the three temperatures and with the use of a cryoprotective agent. Cryopreservation for salvage management is a clinically practical method.     

Intramedullary Cavity as Implantation Site for Bioartifical Pancreas: Preliminary In Vivo Study

Background

The intramedullary cavity is a widely distributed well-vascularized microenvironment capable of sustaining grafts, and is a potential site for islet transplantation. The bone marrow offers sufficient space that may also be suitable for bioartificial pancreas (BAP) implantation.

Objective

To evaluate the feasibility of bone marrow as an implantation site for BAPs.

Materials and Methods

A calcium phosphate cement chamber satisfies the criteria for immunoisolation. Mouse insulinoma cells were suspended with agarose gel and enclosed in a calcium phosphate cement chamber to create a BAP, which was implanted in the intramuscular space in diabetic swine or the intramedullary cavity in diabetic dogs. Blood glucose and C-peptide concentrations were determined perioperatively.

Results

In the swine, the mean ± SD blood glucose concentration decreased from 413 ± 24 mg/dL to 285 ± 47 mg/dL, and was maintained in the range of 285 to 336 mg/dL for 15 days. It increased to 368 to 450 mg/dL after the BAPs were implanted in the intramuscular space. In the dogs, the blood glucose concentration decreased from 422 ± 32 mg/dL to 247 ± 52 mg/dL, and was maintained in the range of 247 to 347 mg/dL after the BAPs were implanted in the intramedullary cavity. The C-peptide concentration increased from 6.1 ± 2.8 pmol/L to 104.7 ± 16.4 pmol/L when the BAPs were implanted in the intramedullary cavity.

Conclusion

This study indicates superior effectiveness of BAPs implanted in the intramedullary cavity compared with the intramuscular space. This observation may be attributed to the greater oxygen tension in the bone marrow. The BAPs in direct contact with the circulatory system receive sufficient blood flow for function and survival. This preliminary study demonstrates that the intramedullary cavity may be an implantation site for BAP transplantation.     

Hand-Assisted Laparoscopic Donor Nephrectomy: Analysis of the Learning Curve in a Training Model In Vivo

Introduction

Hand-assisted laparoscopic donor nephrectomy (HALDN) outcomes are impaired mainly by the risks associated with the learning curve. Considering that practice by in vivo training may reduced this risk, we recently assessed a swine model of HALDN. The aim of this study was to analyze the learning curve of HALDN using an in vivo training model.

Materials and Methods

Ten female white pigs underwent a left and then a right HALDN in the same session for a total of 20 procedures by the same first operator. The HALDN were divided into 2 groups: group A, the first 10 nephrectomies and group B, the latter 10. For each group, we assessed operative times, intraoperative complications, estimated blood loss (EBL), warm ischemia time (WIT), and graft quality.

Results

We observed a significant decrease in operative times among group B. Two right HALDN of group A were converted to open procedures owing to bleeding. The EBL was consequently lower in group B (P < .05); the mean WIT was not significantly different between the 2 study groups. The graft quality was good in 5/8 kidneys evaluated in group A and 9/10 in group B.

Discussion

Standardization of analyzed parameters after a number of procedures, which were comparable to the clinical settings, confirmed the validity of this in vivo training model and its potential utility to allow many transplantation centers to adopt this technique by reducing the risk of the learning curve.     

Successful Transtracheal Lung Ventilation Using a Manual Respiration Valve: An In Vitro and In Vivo Study

Background: Lung ventilation through a thin transtracheal cannula may be attempted in patients with laryngeal stenosis or "cannot intubate, cannot ventilate" situations. It may be impossible to achieve sufficient ventilation if the lungs are spontaneously emptying only through the thin transtracheal cannula, which imposes high resistance to airflow, resulting in dangerous hyperinflation. Therefore, the authors describe the use of a manual respiration valve that serves as a bidirectional pump providing not only inflation but also active deflation of the lungs in case of emergency transtracheal lung ventilation.

Methods: The effectiveness of such a valve was tested in vitro using mechanical lungs in combination with two different cannula sizes and various gas flows. The valve was then tested in five pigs using a transtracheal 16-gauge cannula with three different combinations of inspiratory/expiratory times and gas flows and an occluded upper airway.

Results: In the mechanical lungs, the valve permitted higher minute volumes compared with spontaneous lung emptying. In vivo, the arterial oxygen and carbon dioxide partial pressures increased initially and then remained stable over 1 h (arterial oxygen tension, 470.8 +/- 86.8; arterial carbon dioxide tension, 63.0 +/- 7.2 mmHg). The inspiratory pressures measured in the trachea remained below 10 cm H2O and did not substantially influence central venous and pulmonary artery pressures. Mean arterial pressure and cardiac output were unaffected by the ventilation maneuvers.     

In Vivo Identification,Survival, and Functional Efficacy of Transplanted Hepatocytes in Acute Liver Failure Mice Model by FISH Using Y-Chromosome Probe

Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome–specific probe in a carbon tetrachloride (CCl₄)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 (n = 15), which received mineral oil, served as a negative control. Group-II (n = 15) received CCl₄ (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III (n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl₄ administration. Male Swiss adult mice (n = 15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.     

An Alternative Treatment for the Split Skin-Graft Donor Site

There are a variety of methods employed in the postoperative management of the partial thickness donor site created during harvest of a split thickness skin graft. Each technique may be associated with potential complications of fluid loss, excessive pain, prolonged period for healing and delayed mobility, hypertrophic scarring, undesirable pigment aesthetics, and thin skin poorly resistant to everyday trauma. Thompson, and Converse and Robb-Smith have previously shown improved donor site outcome with the application of thin split skin grafts. Based on these studies, we present a technique that involves 1.5:1 meshing of a split skin graft and dividing it into equal halves so that one half is used to cover the defect and the other half is immediately returned to the donor site. Patients who are elderly, debilitated, or who have thin, poor-quality skin can expect less discomfort, reduction of fluid loss, improved durability and elasticity, and lower incidence of hypertrophic scarring with the proposed donor site regrafting.     

In Vivo CT Quantification of Trabecular Bone Dynamics in Mice after Sciatic Neurectomy Using Monochromatic Synchrotron Radiation

We demonstrated the capability of in vivo synchrotron radiation CT (SRCT) in analyzing short-term changes in trabecular bone architecture (TBA) and the degree of bone mineralization (DBM) in small animals. Mice underwent unilateral sciatic neurectomy (SN) and sham operation on the contralateral side (SO) at 13 weeks of age. In vivo SRCT scans (11.7-μm cubic voxel) were made of both knees 7 and 17 days (group 1, n = 7) or only 17 days (group 2, n = 6) after surgery. In three mice in group 2, one knee was scanned twice on the same day in different orientations for reproducibility testing. Two scan data sets of the tibial proximal metaphysis acquired at different time points (group 1) or at the same time point (group 2) were registered for detecting differences in volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), connectivity density (Conn.D), and mean DBM (mDBM). The reproducibility test showed small errors of <2.5% in the TBA indexes and <3.0% in mDBM, while mismatched bone regions amounted to >25%. In group 1, Tb.Th increased but Tb.N and Conn.D decreased in both SN and SO; BV/TV and mDBM increased only in SO; accordingly, BV/TV, Tb.Th, and mDBM became lower in SN than in SO. No significant interaction between SN and irradiation was found; the SN effects on TBA and DBM were similar between groups 1 and 2, although synchrotron irradiation led to higher Tb.Th and lower Tb.N in group 1. In conclusion, in vivo SRCT has potential use for detecting short-term bone dynamics of small animals.     

In Vivo Follicular Unit Multiplication: Is It Possible to Harvest an Unlimited Donor Supply?

BACKGROUND: Follicular unit extraction is a process of removing one follicular unit at a time from the donor region. The most important limitation of this surgical procedure is a high transection rate. OBJECTIVE: In this clinical study, we have transplanted different parts of transected hair follicle by harvesting with the follicular unit extraction technique (FUE) in five male patients. MATERIALS AND METHODS: In each patient, three boxes of 1 cm(2) are marked at both donor and recipient sites. The proximal one-third, one-half, and two-thirds of 15 hair follicles are extracted from each defined box and transplanted in recipient boxes. The density is determined at 12 months after the procedure. RESULTS: A mean of 3 (range, 2-4) of the proximal one-third, 4.4 (range, 2-6) of the proximal one-half, and 6.2 (range, 5-8) of the proximal two-thirds of the transplanted follicles were observed as fully grown after 1 year. At the donor site, the regrowth rate was a mean of 12.6 (range, 10-14) of the proximal one-third, 10.2 (range, 8-13) of the proximal one-half, and 8 (range, 7-12) of the proximal two-thirds, respectively. CONCLUSION: The survival rate of the transected hair follicles is directly related to the level of transection. Even the transected parts, however, can survive at the recipient site; the growth rate is not satisfactory and they are thinner than the original follicles. We therefore recommend that the surgeon not transplant the sectioned parts and be careful with the patients whose transection rate is high during FUE procedures.     

MMP-2反义寡核苷酸抑制人膀胱癌裸鼠异体移植瘤生长的实验研究

目的：探讨基质金属蛋白酶2（MMP2）反义寡核苷酸对人膀胱癌裸鼠异体移植瘤生长的抑制作用。方法：制备膀胱癌裸鼠移植瘤模型18只,随机分为3组：MMP2反义寡核苷酸（ASODN）治疗组、MMP2正义寡核苷酸（SODN）治疗组及对照组,每组6只。待瘤结节直径≥5mm后,分别在肿瘤细胞接种部位周围及中心皮下注射反义寡核苷酸／阳离子脂质体复合物、正义寡核苷酸／阳离子脂质体复合物和生理盐水。每周2次,连续4周,断颈处死裸鼠,计算肿瘤体积,称量瘤重。常规切片,HE染色,观察组织学形态并进行病理学评估。应用免疫组织化学技术（SP法）检测各组移植瘤组织中PCNA蛋白表达。结果：与对照组瘤重（7．49±0．53）g比较,ASODN组瘤重（4．18±0．53）g明屁降低（P〈0．01）;ASODN组、正义寡核苷酸（SODN）组抑瘤率分别为44．19％、8．41％（P〈0．01）;ASODN组、对照组增殖指数（PI值）分别为27．63％、88．39％,抑制率为60．76％（P〈0．01）;ASODN组瘤体病理学特征改善。结论：MMP-2反义寡核苷酸能抑制裸鼠体内移植瘤生长,通过反义寡核苷酸下调MMP-2的表达,降低癌细胞的增殖活性,有效逆转肿瘤的恶性表型。为膀胱癌的基因治疗提供了实验依据。     

前列腺癌细胞株Du145裸鼠胫骨部骨肿瘤转移模型的建立

目的:建立一种裸鼠前列腺癌骨转移模型,观察肿瘤局部生长和远处转移情况,并进一步探讨其应用价值。方法:9只6周龄雄性BALB/C裸鼠麻醉后,将前肢及左后肢固定,以1 m l TB针筒-29G针头从胫骨头关节窝,刺入骨髓腔缓缓注入人前列腺癌Du145细胞悬液30μl(约5×106个细胞)至骨髓腔,以建立前列腺癌骨转移模型。观察裸鼠生命体征变化。于濒死状态下处死,取右后肢及其附近淋巴结、肺脏和肝脏等标本,用40 m l/L甲醛固定、石蜡包埋、苏木精-伊红染色后显微镜下观察。结果:9只裸鼠中有6只模型建立成功。接种后平均48 d裸鼠出现跛行现象,右后肢胫骨部可触及米粒大小的包块,质硬,进一步发展至小鼠行走障碍。55 d后出现恶病质,解剖后发现右后肢胫骨部骨质疏松,局部有“腐肉”样组织突出髓腔,填塞肌肉间隙,病理证实为肿瘤组织。肝脏泛黄、被膜皱缩,镜下呈急性重型肝炎的病理改变。结论:胫骨内注射细胞悬液制作裸鼠前列腺癌骨转移模型较好地模拟了人前列腺癌在骨骼微环境中的生长及转归情况,是研究前列腺肿瘤骨转移的适宜模型。     

Aortic Customize: An In Vivo Feasibility Study of a Percutaneous Technique for the Repair of Aortic Aneurysms Using Injectable Elastomer

ObjectiveThis study aimed to test a percutaneous technique for aneurysm-sac filling by means of in situ polymerisation in an in vivo model.DesignAortic Customize is a new endovascular treatment concept for aortic aneurysms: a non-cross-linked liquid elastomer is injected to fill the aneurysm sac around a balloon-catheter. With this method, a compliant elastomer mould with a patent lumen is created.MaterialThe formulation used in the experiments consisted of a two-component addition-cure liquid-silicone formulation, based on vinyl-terminated polydimethylsiloxane (PDMS).MethodsThe concept of aneurysm-sac filling was tested in vivo in porcine experiments (n = 3).ResultsIn vivo porcine experiments with the sac-filling application showed successful exclusion of the created aneurysms with patent lumens and absence of endoleaks. The aneurysms were excluded successfully in the in vivo model, injecting elastomer through a 7-French catheter, filling up the entire aneurysm sac.ConclusionsThese in vivo experiments demonstrate that the principle of aneurysm-sac filling by means of in situ curing is feasible, excluding the aneurysm and creating a new lumen. Further long-term animal experiments must be done prior to consideration of clinical application.     

Comparison of Ex Vivo and In Vivo Injection of Blue Dye in Sentinel Lymph Node Mapping for Colorectal Cancer

Background  The technique of sentinel lymph node (SLN) mapping in patients with colorectal cancer varies between reports, and the optimal method has not been established. The purpose of this study was to determine the optimal injection technique for SLN mapping. Methods  Sixty-nine consecutive patients who underwent curative surgery for colorectal cancer were enrolled. The SLNs was identified intraoperatively by subserosal blue dye injection (in vivo) or by submucosal injection after standard colectomy (ex vivo). If negative by conventional hematoxylin and eosin staining analysis, all lymph nodes, SLNs and non-SLNs, were subjected to further analysis by multi-level section and immunohistochemical examination. Results  The in vivo and ex vivo injected groups were similar in demographic character, tumor size, and histological grade. The mean number of SLNs identified was 2.3 in the in vivo group and 2.6 in the ex vivo group (p = 0.192). The detection rate of SLNs by blue dye injection was somewhat higher in the ex vivo group than in the in vivo group: 90.6 vs. 81.1% (p = 0.219). The false-negative rate was 23.5% for the in vivo group and 13.3% for the ex vivo group (p = 0.392). The upstaging rate, which was 18.5% overall, was similar in both groups (p = 0.538). Conclusions  These findings suggest that ex vivo blue dye injection is an effective alternative to in vivo injection for identifying SLNs in patients with colorectal cancer. Because of its simplicity and applicability in routine clinical settings, further investigation of the ex vivo mapping technique is warranted. Research was supported by the Chung-Ang University Research Grants in 2008.     

Prevention of Fat Embolism in Fat Injection for Gluteal Augmentation,Anatomic Study in Fresh Cadavers

Introduction: Liposuction is a popular surgical procedure. As in any surgery, there are risks and complications, especially when combined with fat injection. Case reports of fat embolism have described a possible explanation as the puncture and tear of gluteal vessels during the procedure, especially when a deep injection is planned. Methods: A total of 10 dissections were performed in five fresh cadavers. Each buttocks was divided into four quadrants. We focused on the location where the gluteal vessels enter the muscle and the diameter of the vessels. Colorant at two different angles was injected (30° and 45°). We evaluated the relation of the colorant with the main vessels. Results: We found two perforators per quadrant. The thickness of the gluteal muscle was 2.84 ± 1.54 cm. The area under the muscle where the superior gluteal vessels traverse the muscle was located 6.4 ± 1.54 cm from the intergluteal crease and 5.8 ± 1.13 cm from the superior border of the muscle. The inferior gluteal vessels were located 8.3 ± 1.39 cm from the intergluteal crease and 10 ± 2.24 cm from the superior border of the muscle. When we compared the fat injected at a 30° angle, the colorant stayed in the muscle. Using a 45° angle, the colorant was in contact with the superior gluteal artery and the sciatic nerve. No puncture or tear was observed in the vessels or the nerve. Conclusions: The location where the vessels come in contact with the muscle, which can be considered for fat injection, were located in quadrants 1 and 3. A 30° angle allows for an injection into the muscle without passing into deeper structures, unlike a 45° injection angle.     

Immunosuppressive Effects of Emodin: An In Vivo and In Vitro Study

Objective

We sought to investigate the immunosuppressive effects of emodin and its potential in vivo and in vitro mechanisms.

Methods

In vitro immunosuppressive effects of emodin were analyzed by its ability to suppress the response of human peripheral blood mononuclear cells to phytohemagglutinin (PHA) and to mixed lymphocyte culture (MLC). We examined changes in interleukin (IL)-2 and IL-4 in within MLC supernates. The in vivo immunosuppressive effects of emodin were analyzed using a skin transplantation model in mice. We also investigated the mean survival time (MST) and plasma IL-2 levels.

Results

In vitro experiments: Responses of mononuclear cells to PHA and MLC were suppressed by emodin treatment. Decreased production of IL-2 along with promoted secretion of IL-4 was also observed by emodin treatment during MLC. In vivo experiments: The emodin-treated group showed prolonged MST of skin grafts and decreased serum IL-2 production.

Conclusions

Emodin showed immunosuppressive activities both in vivo and in vitro. The potential immunosuppressive mechanism of emodin's may be suppression of lymphocyte proliferation and influences on cytokines.     

Fat Transplantation Using Fresh Versus Frozen Fat: A Side-by-Side Two-Hand Comparison Pilot Study

BACKGROUND: Autologous fat in both fresh and frozen forms has been used for many years as a filler for various dermatologic conditions. However, it is not clear whether fat that has been frozen survives as well as, and gives aesthetic results similar to, fresh fat. The efficacy of frozen fat has been debated in the literature. OBJECTIVE: To evaluate the clinical aesthetic appearance and longevity of fresh fat versus frozen fat in a side-by-side two-hand comparison in the same patient. METHODS: Ten patients underwent fat augmentation on their hands, utilizing 10 cc of fresh fat in one hand and 10 cc of frozen fat in the contralateral hand within 17 days of fresh fat placement. Follow-up evaluation was conducted at 1, 3, and 5 months in a randomized, double-blind comparison study. Physician-determined aesthetic preference, prominence of veins, and depth of metacarpal spaces were evaluated. Photographs were taken of both hands during each patient follow-up visit. RESULTS: All three areas of physician-assessed gradation: aesthetics, vein prominence, and depth of metacarpal space were superior for the hand injected with frozen fat at 1-, 3-, and 5-month follow-up visits. CONCLUSIONS: This pilot study supports the use of autologous frozen fat for equivalent to improved results regarding longevity and aesthetic appearance versus fresh fat at 1, 3, and 5 months for fat augmentation of aging hands.     

Quantifying Massive Allograft Healing of the Canine Femur In Vivo and Ex Vivo: A Pilot Study

Background

Allograft integration in segmental osseous defects is unpredictable. Imaging techniques have not been applied to investigate angiogenesis and bone formation during allograft healing in a large-animal model.

Questions/purposes

We used dynamic contrast-enhanced (DCE)-MRI and cone beam (CB)-CT to quantify vascularity and bone volume in a canine femoral allograft model and determined their relationship with biomechanical testing and histomorphometry.

Methods

Femoral ostectomy was performed in three dogs and reconstructed with a 5-cm allograft and compression plate. At 0.5, 3, and 6 months, we performed DCE-MRI to quantify vascular permeability (Ktrans) and perfused fraction and CB-CT to quantify bone volume. We also performed posteuthanasia torsional testing and dynamic histomorphometry of the grafted and nonoperated femurs.

Results

DCE-MRI confirmed the avascular nature of allograft healing (perfused fraction, 2.08%–3.25%). CB-CT demonstrated new bone formation at 3 months (26.2, 3.7, and 2.2 cm³) at the graft-host junctions, which remodeled down at 6 months (14.0, 2.2, and 2.0 cm³). The increased bone volume in one subject was confirmed with elevated Ktrans (0.22) at 3 months. CB-CT-identified remodeled bone at 6 months was corroborated by histomorphometry. Allografted femurs recovered only 40% of their strength at 6 months.

Conclusions

CB-CT and DCE-MRI can discriminate differences in angiogenesis and bone formation in the canine allograft model, which has potential to detect a small (32%) drug or device effect on biomechanical healing with only five animals per group.

Clinical Relevance

These radiographic tools may have the potential to assess adjuvant effects on vascular invasion and new bone formation after segmental allograft transplantation.