前脂肪细胞组织工程学的初步探讨

目的探讨胶原-透明质酸海绵支架与前脂肪细胞复合培养形成工程化脂肪组织的可行性。方法切取成年女性腹部皮下脂肪组织，经体外分离培养前脂肪细胞，与胶原-透明质酸支架混合，移植到裸鼠体内，未混合细胞的支架作为对照组。移植后4周和8周取材，对所取标本进行大体观察及厚度、高度测量和免疫组织化学检测。结果初步证实复合物植入后能形成黄色脂肪样组织。有新生血管形成，免疫组织化学检测证实脂肪细胞的存在及良好分布。结论胶原一透明质酸海绵支架可以作为组织工程技术应用研究中形成脂肪组织的支架，移植到裸鼠体内，在其皮下能形成脂肪样组织。     

人发角蛋白材料植入修复皮下脂肪缺损的实验观察

目的 探讨人发角蛋白材料植入皮下脂肪缺损处能否形成新生脂肪组织,旨在为皮下肿物切除术后即时填充修复及矫正陈旧性体表凹陷畸形提供一条新的途径. 方法 采用西藏小型猪3只,在猪脊柱两侧皮下各形成脂肪缺损8处,直径约1.5 cm.将人发角蛋白材料制成直径约1.5 cm球状植入一侧缺损处,另一侧脂肪缺损作为空白对照.采用组织学方法观察术后不同时间人发角蛋白材料降解及新生脂肪组织生成情况. 结果 人发角蛋白材料植入后2周,可见结缔组织及微血管从材料孔隙长入;植入后4周,人发角蛋白基本降解吸收,可见材料碎屑及异物肉芽肿,其周围见成簇的新生脂肪细胞;植入后6周,人发角蛋白材料完全降解,异物肉芽肿也随之消失,代之以新生脂肪组织,其体积与当初植入的人发角蛋白材料体积接近;植入后10周,新生脂肪组织中的纤维成分减少,接近周围正常脂肪组织. 结论 人发角蛋白材料植入皮下脂肪组织缺损处,可在原位形成足够体积的新生脂肪组织,并逐渐塑形,从而在组织结构上接近正常脂肪组织.     

Engineering of Adipose Tissue by Injection of Human Preadipocytes in Fibrin

Background Despite efforts of plastic surgeons in recent years to discover new alternatives, the techniques currently used for restoration of soft tissue defects still have disadvantages. The gold standard for soft tissue reconstruction remains autologous pedicled/free tissue transfer. This technique often results in high rates of operative morbidity and donor site deformity. Results obtained by autologous fat tissue transfer usually are disappointing because of a high graft resorption rate with unpredictable outcomes. Different tissue engineering approaches have been used in the past to generate adipose tissue. However, long-term results in terms of volume persistence have been disappointing. Methods In this study, different concentrations of undifferentiated human preadipocytes in fibrin were injected into athymic nude mice (n = 8). Mice that had fibrin injection without cells served as control subjects (n = 8). The specimens were explanted after 1, 2, 6, and 9 months, with subsequent qualitative and quantitative analysis of adipose tissue formation by histologic and image analysis. Results Within the first 4 weeks after initial volume reduction of the implants, the volume and shape of the implants with preadipocytes remained stable. The implants without cells were completely resorbed within 3 weeks. Histologic analysis demonstrated generation of stable adipose tissue with no signs of an inflammatory response or evidence of tissue necrosis in the implants containing preadipocytes. The best results were obtained after implantation of 30 million preadipocytes. Adipose tissue formation was not observed in the control group. Conclusions The findings demonstrate that long-term stable adipose tissue can be engineered in vivo by simple injection of human preadipocytes using fibrin as a carrier material. After further investigation, this approach may represent an alternative to the techniques currently used for soft tissue restoration. Presented at the 36th Conference of the Association of German Plastic Surgeons (DGPRA) in Munich, Germany, September 2005, and at the 8th Conference of the Association of French Aesthetic and Plastic Surgeons (SOFCEP) in Paris, France, June 2005     

非诱导脂肪源性干细胞对修复全层透明软骨创伤的可行性研究

目的探讨源自分布广泛的脂肪组织的干细胞的体内分化潜能及非诱导修复全层透明软骨缺损的效果。方法体外切取脂肪组织并分离培养脂肪源性干细胞（ADSCs）。随机将36只新西兰大白兔分为三组,混合有藻酸钙凝胶的ADSCs用来填充髌股关节的全层透明软骨损伤,凝胶修复或未做治疗组作为对照组。4周和12周后对重建组织进行大体和光镜、电镜下观察,组织学分析和定量计分也用于检测结果。结果ADSCs重建的组织白色质韧,完全充填缺损处,表面光整与周围软骨连接,修复组织的微观结构与软骨相似,含有更多的细胞和规则的基质纤维,基质有甲苯胺蓝异染性,优于其他各组。透射电镜可见大量胶原纤维环绕细胞周围。凝胶组和对照组修复组织为薄层纤维组织。修复效果评分的统计分析显示实验组在各时问点上与其他组相比有统计学差异（P〈0.01。结论这些结果表明源自成熟脂肪而未经诱导的干细胞拥有修复软骨创伤的能力,组织显示为透明样,产生生物学可行性结果。     

经RNM诱导的前脂肪细胞构建组织工程化脂肪组织

目的:应用基因工程和组织工程的原理和方法,通过改造人前体脂肪细胞,提高其活性后,于裸鼠背部皮下构建组织工程化脂肪组织.方法:采用原代消化细胞培养法培养出前脂肪细胞,利用RNAi技术,诱导前脂肪细胞内的一个重要凋亡调控基因Bax基因表达沉默,然后接种到聚乳酸一乙醇酸共聚物(PLGA)支架上,种植在20只雌性裸鼠皮下,对获得的脂肪组织进行组织学评价.结果:经过改造后的前脂肪细胞活性良好,术后裸鼠全部成活,前脂肪细胞能够在支架上良好的存活,生长和增殖.结论:经过改造后的前脂肪细胞具有良好的活性,并且PLGA-前脂肪细胞复合体在裸鼠体内可形成脂肪细胞,值得进一步研究,以探索治疗软组织缺损的新途径.     

Uninduced adipose-derived stem cells repair the defect of full-thickness hyaline cartilage

Objective: To testify the effect of the stem cells derived from the widely distributed fat tissue on repairing full-thickness hyaline cartilage defects.Methods: Adipose-derived stem cells (ADSCs) were derived from adipose tissue and cultured in vitro.Twentyseven New Zealand white rabbits were divided into three groups randomly.The cultured ADSCs mixed with calcium alginate gel were used to fill the full-thickness hyaline cartilage defects created at the patellafemoral joint,and the defects repaired with gel or without treatment served as control groups.After 4,8 and 12 weeks,the reconstructed tissue was evaluated macroscopically and microscopically.Histological analysis and qualitative scoring were also performed to detect the outcome.Results: Full thickness hyaline cartilage defects were repaired completely with ADSCs-derived dssue.The result was better in ADSCs group than the control ones.The microstructure of reconstructed tissue with ADSCs was similar to that of hvaline cartilage and contained more cells and regular matrix fibers,being better than other groups.Plenty of collagen fibers around cells could be seen under transmission electron microscopy.Statistical analysis revealed a significant difference in comparison with other groups at each time point(t=4.360,P＜0.01).Conclusion: Thcse results indicate that stem cells derived from mature adipose without induction possess the ability to repair cartilage defects     

Comparison of different fabrication techniques for human adipose tissue engineering in severe combined immunodeficient mice

Adipose tissue engineering has been advocated for soft‐tissue augmentation and for the treatment of soft tissue defects. The efficacy in terms of persistence of the engineered fat is, however, not yet understood and could depend on the nature of fabrication and application. The high metabolic demand of adipose tissue also points to the problem of vascularization. Endothelial cell (EC) cotransplantation could be a solution. Human adipose tissue‐derived stromal cells were seeded on collagen microcarriers and submitted to adipogenic differentiation (“microparticles”). In a first run of experiments, these microparticles were implanted under the skin of severe combined immunodeficient (SCID) mice (n = 45) with and without the addition of human umbilical vein ECs (HUVECs). A group of carriers without any cells served as control. In a second run, adipose tissue constructs were fabricated by embedding microparticles in fibrin matrix with and without the addition of HUVEC, and were also implanted in SCID mice (n = 30). The mice were sacrificed after 12 days, 4 weeks, and 4 months. Mature adipose tissue, fibrous tissue, and acellular regions were quantified on whole‐specimen histological sections. The implantation of microparticles showed a better sustainment of tissue volume and a higher degree of mature adipose tissue compared with adipose tissue constructs. Immunohistology proved obviously perfused human tissue‐engineered vessels. There was a limited but not significant advantage in EC cotransplantation after 4 weeks in terms of tissue volume. In groups with EC cotransplantation, there were significantly fewer acellular/necrotic areas after 4 weeks and 4 months. In conclusion, the size of the implanted tissue equivalents is a crucial parameter, affecting volume maintenance and the gain of mature adipose tissue. EC cotransplantation leads to functional stable vascular networks connecting in part to the host vasculature and contributing to tissue perfusion; however, the long‐term benefit depends on additional basic conditions that need further research.     

Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats

Muscle and hepatic insulin resistance are two major defects of non-insulin-dependent diabetes mellitus. Dietary factors may be important in the etiology of insulin resistance. We studied progressive changes in the development of high-fat-diet-induced insulin resistance in tissues of the adult male Wistar rat. In vivo insulin action was compared 3 days and 3 wk after isocaloric synthetic high-fat or high-starch feeding (59 and 10% cal as fat, respectively). Basal and insulin-stimulated glucose metabolism were assessed in the conscious 5- to 7-h fasted state with the euglycemic clamp (600 pM insulin) with a [3-3H]-glucose infusion. Fat feeding significantly reduced suppressibility of hepatic glucose output by insulin after both 3 days and 3 wk of diet (P less than 0.01). However, a significant impairment of insulin-mediated peripheral glucose disposal was only present after 3 wk of diet. Further in vivo [3H]-2-deoxyglucose uptake studies supported this finding and demonstrated adipose but not muscle insulin resistance after 3 days of high-fat feeding. Muscle triglyceride accumulation due to fat feeding was not significant at 3 days but had doubled by 3 wk in red muscle (P less than 0.001) compared with starch-fed controls. By 3 wk, high-fat-fed animals had developed significant glucose intolerance. We conclude that fat feeding induces insulin resistance in liver and adipose tissue before skeletal muscle with early metabolic changes favoring an oversupply of energy substrate to skeletal muscle relative to metabolic needs. This may generate later muscle insulin resistance.     

Potential of Fortified Fibrin/Hyaluronic Acid Composite Gel as a Cell Delivery Vehicle for Chondrocytes

Numerous treatment methods have been applied for use in cartilage repair, including abrasion, drilling, and microfracture. Although chondrocyte transplantation is the preferred treatment, it has some shortcomings, such as difficulty of application (large and posterior condylar regions), poor chondrocyte distribution, and potential cell leakage from the defect region. The cell delivery system of the tissue engineering technique can be used to overcome these shortcomings. We chose fibrin/hyaluronan (HA) composite gel as an effective cell delivery system to resolve these issues. Both components are derived from natural extracellular matrix. In the first trial, fortified fibrin/HA composite gels with rabbit chondrocytes were tested by implantation in nude mice. At 4 weeks, glycosaminoglycan contents in the fibrin/HA composite (0.186 ±  0.006 mg/mg) were significantly higher than those in the presence of fibrin alone (0.153 ± 0.017 mg/mg). As a next step, we applied the fibrin/HA composite gel to animal cartilage defects using full thickness cartilage defect rabbit models. The fibrin/HA composite gel with rabbit chondrocytes (allogenic) was implanted into the experimental group, and the control group was implanted with the fibrin/HA composite gel alone. Implanted chondrocytes with the fibrin/HA composite showed improved cartilage formation. In conclusion, the key to successful regeneration of cartilage is to provide the repair site with a sufficient supply of chondrogenic cells with a suitable delivery vehicle to ensure maximal differentiation and deposition of the proper extracellular matrix. This study suggests the feasibility of tissue-engineered cartilage formation using fibrin/HA composite gel.     

Osteochondral regeneration using constructs of mesenchymal stem cells made by bio three‐dimensional printing in mini‐pigs

Osteoarthritis is a major joint disease that has been extensively investigated in humans and in model animals. In this study, we examined the regeneration of articular cartilage and subchondral bone using artificial scaffold‐free constructs composed of adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) created using bio three‐dimensional (3D) printing with a needle‐array. Printed constructs were implanted into osteochondral defects created in the right femoral trochlear groove of six mini‐pigs, using femoral defects created in the left femurs as controls. Repair within the defects was evaluated at 3 and 6 months post‐implantation using computed tomography (CT) and magnetic resonance (MR) imaging. The radiolucent volume (RV, mm³) in the defects was calculated using multi‐planar reconstruction of CT images. MR images were evaluated based on a modified 2D‐ MOCART (magnetic resonance observation of cartilage repair tissue) grading system. Gross and microscopic pathology were scored according to the ICRS (International Cartilage Repair Society) scale at 6 months after implantation. The percentage RV at 3 months postoperation was significantly lower in the implanted defects than in the controls, whereas total scores based on the MOCART system were significantly higher in the implanted defects as compared with the controls. Although there were no statistical differences in the gross scores, the average histological scores were significantly higher in the implanted defects than in the controls. To our knowledge, this is the first report to suggest that artificial scaffold‐free 3D‐printed constructs of autologous AT‐MSCs can be aid in the osteochondral regeneration in pigs. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1398–1408, 2019.     

Clinical and Experimental Study of Autologous Fat Grafting After Processing by Centrifugation and Serum Lavage

Background  This clinical and experimental study compared adipose tissue transplant behavior after two different techniques of purifying: centrifugation at 3400 rpm for 3 min and serum lavage without centrifugation. Methods  Clinical evaluation was performed under standardized conditions for lipofilling on a series of 51 female patients, intentionally selected to have similar characteristics and assigned to two groups based on the method of processing. Experimentally, a culture system in diffusion chambers with vitaline membranes was designed to mimic the behavior and to study the morphology of the adipose tissue used for autografting. Survival, structure, and proliferation of the adipose cells in vitro were examined by classical histologic H&E staining and immunohistochemistry for leptin and cyclin D1. Results  The main differences encountered experimentally were the presence of a greater amount of preadipocytes in the noncentrifuged adipose tissue cultures and more distinctly expressed cell proliferation. The postoperative clinical results favored of the serum lavage purifying technique. Conclusion  Our data suggest that with transplantation of noncentrifuged adipose tissue more active preadipocytes are applied which could possibly lead to better potential chances of survival and even de novo development of fat.     

The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels.

Autologous chondrocyte implantation (ACI) is the most promising surgical treatment for large full thickness knee joint articular cartilage (AC) defects where cells from healthy non-weight bearing area AC are multiplied in vitro and implanted into such defects. In the routine surgical procedure for symptomatic knee full thickness AC defects, damaged AC surrounding the edge and the base of such defects is usually debrided and discarded. The purpose of this study was to examine if chondrocytes from this 'debrided' AC can proliferate, synthesize a cartilage specific matrix and thus can be used for ACI. METHODS: Biopsies were retrieved from 12 patients (debrided articular cartilage: DAC, aged 35-61) and from two autopsies (normal articular cartilage: NAC, aged 21 and 25). Chondrocytes were isolated, seeded at low density in type I collagen gels and as monolayer cultures for 4 weeks without passage. RESULTS: After 4 weeks cultures in type I collagen gels, cell proliferation from DAC (18.34 +/- 1.95 fold) was similar to cells from NAC (11.24 +/- 1.02 fold). Syntheses of proteoglycan and collagen in DAC were also similar to NAC. Newly synthesized matrices in gel cultures consisted predominantly of type II collagen as shown by immuno-labelling and SDS-PAGE followed by fluorography. Chondrocytes from 'debrided human AC' cultured at low density in type I collagen gels may be used for the ACI procedure as they provide sufficient viable cell numbers for ACI and maintain their chondrocyte phenotype as they synthesize a cartilage-like matrix.     

Engineered adipose tissue formation enhanced by basic fibroblast growth factor and a mechanically stable environment

Engineered adipose tissue can be used in plastic and reconstructive surgery to augment soft tissue lost due to mastectomy or lumpectomy. The three-dimensional space provided by a scaffold capable of withstanding in vivo compressive forces and neovascularization may promote engineered adipose tissue formation. The objective of this study was to determine whether voluminous adipose tissue can be engineered by combining a mechanically stable environment with basic fibroblast growth factor (bFGF). Mechanical support structures, fabricated from biodegradable synthetic polymers, were placed into subcutaneous pockets of athymic mice. Human preadipocytes, containing fibrin matrix, with (group 1) or without (group 2) bFGF were injected into the space created by the support structures. Additionally, human preadipocytes containing fibrin matrix, with (group 3) or without (group 4) bFGF, were injected into subcutaneous spaces without support structures. Six weeks after implantation, the original implant volume was approximately maintained in groups 1 and 2, whereas groups 3 and 4 showed significant implant shrinkage. Adipogenesis and angiogenesis were more extensive in the group 1 than any other group. The fraction of human nuclear antigen-positive adipocytes in the implant was highest in group 1. Mouse adipocyte-specific genes were also expressed in the implants, again at the highest levels in group 1. Implanted preadipocyte apoptosis was significantly reduced in the groups treated with bFGF (groups 1 and 3) as opposed to those without (groups 2 and 4). This study demonstrates that combining a mechanically stable environment with bFGF can promote voluminous adipose tissue regeneration. This adipogenesis was likely promoted by the mechanically stable three-dimensional space, enhanced neovascularization, implanted cell survival, and host adipogenic cell migration. The method described in this study could be useful to augment adipose tissue used in plastic and reconstructive surgery.     

Tissue engineering of human fat for soft tissue augmentation: in vitro results

. In our study we investigated whether human preadipocytes from human fat derived from routine abdominoplasty were suitable for the in vitro cultivation of fat. We could isolate and culture preadipocytes from patients of different ages and stimulate the cultured cells to differentiate into adipocytes and accumulate fat. We noticed age-related differences in the growth rate and differentiation capability into mature adipocytes. We believe that in the future a cell suspension of preadipocytes from a patient amplified in tissue culture might provide a relatively unlimited amount of injectable autologous transplant to fill soft tissue defects.     

Fibroblast growth factor 1: a key regulator of human adipogenesis

Obesity, with its related problems, is recognized as the fastest growing disease epidemic facing the world, yet we still have limited insight into the regulation of adipose tissue mass in humans. We have previously shown that adipose-derived microvascular endothelial cells (MVECs) secrete a factor(s) that increases proliferation of human preadipocytes. We now demonstrate that coculture of human preadipocytes with MVECs significantly increases preadipocyte differentiation, evidenced by dramatically increased triacylglycerol accumulation and glycerol-3-phosphate dehydrogenase activity compared with controls. Subsequent analysis identified fibroblast growth factor (FGF)-1 as an adipogenic factor produced by MVECs. Expression of FGF-1 was demonstrated in MVECs but not in preadipocytes, while preadipocytes were shown to express FGF receptors 1-4. The proliferative effect of MVECs on human preadipocytes was blocked using a neutralizing antibody specific for FGF-1. Pharmacological inhibition of FGF-1 signaling at multiple steps inhibits preadipocyte replication and differentiation, supporting the key adipogenic role of FGF-1. We also show that 3T3-L1 cells, a highly efficient murine model of adipogenesis, express FGF-1 and, unlike human preadipocytes, display no increased differentiation potential in response to exogenous FGF-1. Conversely, FGF-1-treated human preadipocytes proliferate rapidly and differentiate with high efficiency in a manner characteristic of 3T3-L1 cells. We therefore suggest that FGF-1 is a key human adipogenic factor, and these data expand our understanding of human fat tissue growth and have significant potential for development of novel therapeutic strategies in the prevention and management of human obesity.     

人前脂肪细胞培养及增殖与分化模型的建立

目的 更深入地研究人体脂肪组织增生和肥大的生物学特征。方法 选用成人的纯脂肪粒,采用“贴壁－天花板”培养法培养出梭形细胞。结果 培养出的梭形细胞成分均一,增殖旺盛,分化率高,经形态学动态变化,生长、甘油磷酸氢酶活性变化,油红Ｏ脂肪染以及对胰岛素和地塞米松反应的测定,证明是功能活跃的前脂肪细胞,并在体外了其增殖、肥大的全过程。结论在成熟的脂肪中着功能活跃的可调控组分。这是在国内首先建立的一种新的简便     

Aspiration lipectomy and adipose tissue injection pathophysiologic commentary

Summary The subcutis, made up of connective and adipose tissue, may consist of one or two layers, depending on the part of the body. The superficial layer, up to 10 mm in thickness, has the function of giving contour. In localizations where one typically sees relatively large fat deposits a padding layer is added to the contour layer, and this may be centimeters in thickness. Aspiration lipectomy should be limited to reducing the padding layer. The advancing cannula perforates the supportive structures in the subcutis and breaks up the fat lobules. Analysis of the aspirated material suggests that residues of fat lobules remain adhering to the chamber walls; it may be assumed that they necrose and are removed by the mechanisms of foreign body reaction. Some stiffening of the reduced subcutis due to fibroproductive processes appears to be inevitable. Implantation of fat fragments obtained through lipectomy offers little biological advantage; at the same time one must expect extensive necrosis of the implanted tissue. With fat cell suspensions, failure to heal seems to be due to inadequate restoration of the capillary bed. The use of cultured preadipocytes to build up adipose tissue is an attractive idea, but the method is still at the experimental stage. Presented on the 19th Congress of the German Society for Plastic Surgery in Düsseldorf, September 27–30, 1989     

Downregulation of electron transport chain genes in visceral adipose tissue in type 2 diabetes independent of obesity and possibly involving tumor necrosis factor-alpha

Impaired oxidative phosphorylation is suggested as a factor behind insulin resistance of skeletal muscle in type 2 diabetes. The role of oxidative phosphorylation in adipose tissue was elucidated from results of Affymetrix gene profiling in subcutaneous and visceral adipose tissue of eight nonobese healthy, eight obese healthy, and eight obese type 2 diabetic women. Downregulation of several genes in the electron transport chain was the most prominent finding in visceral fat of type 2 diabetic women independent of obesity, but the gene pattern was distinct from that previously reported in skeletal muscle in type 2 diabetes. A similar but much weaker effect was observed in subcutaneous fat. Tumor necrosis factor-alpha (TNF-alpha) is a major factor behind inflammation and insulin resistance in adipose tissue. TNF-alpha treatment decreased mRNA expression of electron transport chain genes and also inhibited fatty acid oxidation when differentiated human preadipocytes were treated with the cytokine for 48 h. Thus, type 2 diabetes is associated with a tissue- and region-specific downregulation of oxidative phosphorylation genes that is independent of obesity and at least in part mediated by TNF-alpha, suggesting that impaired oxidative phosphorylation of visceral adipose tissue has pathogenic importance for development of type 2 diabetes.     

The Fat Fraction Percentage of White Adipose Tissue at various Ages in Humans: An Updated Review

We recently reported the fat fraction percentage of white adipose tissue in adolescents and adults measured by the water-fat separation method, but there was limited discussion about the change in adipose tissue fat fraction with growth. The purpose of this updated review was to examine the fat content of white (subcutaneous) adipose tissue during the process from birth to adulthood by adding the latest available data. A relevant database was searched through November 2020. Nineteen studies were included. We found that calculated mean values of fat fraction percentage in white adipose tissue were 72.2% in neonates, 87.2% in children, and 87.4% in adults. In contrast, fat fraction percentage of truncal white adipose tissue in the fetuses was from 10% to 24% (29 and 34 wk of gestational age, respectively). Our results suggest that the fat fraction percentage of white adipose tissue may not undergo large changes during the process from birth to adulthood (neonates = 72.2%, children = 87.2%, adults = 87.4%), which was different from the results of a study utilizing a biopsy. The mean value and range of fat fraction percentages for children over 7 years old were especially similar to adults. Further, the fat fraction percentage for neonates was relatively close to the results of children and adults. At the moment, the characteristics of the changes in fat fraction percentage of adipose tissue from birth to preschool children are unclear and future research is needed to clarify this issue.