血管基质片段细胞促进游离脂肪移植后再生机制的实验研究
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摘要
研究背景和目的
因外伤、疾病、衰老和先天畸形所造成的软组织缺损一直是整形外科学面临的常见问题。为解决上述问题,胶原注射、真皮移植、人工合成材料植入等软组织填充方法被广泛应用于临床。1889年,Vander Meulen首先报道了脂肪组织的自体移植,至今已有百余年的历史。自体脂肪移植具有的低免疫原性、供区低损伤、操作简便等优点使得这项技术自上世纪30年代起便被广泛应用于整形外科。
尽管脂肪移植效果明显,但长期移植的效应难以控制,其高吸收率、低存活率、多并发症,限制了它在临床中的应用。游离脂肪移植物也面临着脂肪液化、移植物吸收、纤维组织替代等问题,它使得移植物随时间推移的最终保存量仅为最初体积的40-60%。研究发现,在移植早期,脂肪移植物缺乏有效的血液供应而处于急性缺血缺氧状态。在移植物与宿主建立充分的血供之前,脂肪组织只能靠周围组织液的浸润和渗透来维持营养供应,这时中央部分的脂肪组织已经由于缺血缺氧时间过长,发生了坏死、液化。由此可见,在自体颗粒脂肪移植后的转归过程中,移植早期建立及时、充分的血供,移植物及时地再血管化是影响其成活体积的关键。因此,如何促进移植物在短时间内血管化以提高移植脂肪的存活率成为了当前研究的焦点。
近年来随着人们对干细胞研究的不断深入,利用干细胞移植疗法来促进移植物的再血管化,为有效促进移植组织再血管化展示了美好的前景。Makoto等研究证明脂肪组织来源的干细胞(Adipose derived stem cells, ASCs)可促进大鼠后肢局部缺血模型的再血管化,缺血的肌肉组织含有自ASCs转化的血管内皮细胞。本课题组前期的研究发现,体外培养的人ASCs混合脂肪颗粒移植于裸鼠皮下,促进新生血管形成,显著提高脂肪移植物的存活率。然而,临床应用自体培养的ASCs有其局限性,体外培养获取自体ASCs显然无法完全满足临床需要。体外扩增耗时长,至少需要2-3周的时间,这就意味者接受脂肪移植的患者需要二期手术。不仅如此,体外培养操作繁琐,易污染,安全性低,对设施及器材要求严格,费用较高,临床应用受限制。
既然ASCs来源于脂肪中血管基质层细胞(stromal vascular fraction cells, SVFs),且SVFs可通过新鲜分离获得,那SVFs是否能替代ASCs成为促进游离移植脂肪早期血管化的种子细胞呢?2008年,日本学者Kotaro Yoshimura第一个报道了脂肪中SVFs辅助游离脂肪移植的临床应用,研究证实与单纯脂肪移植相比,SVFs辅助的脂肪移植能明显降低脂肪的纤维化、液化,能显著提高脂肪移植的细胞存活率。A. van Dijk等人发现将SVFs静脉注射入心肌梗死小鼠体内,可显著促进缺血心肌组织的再血管化。最近还有研究指出,SVFs可通过促进小鼠游离皮瓣再血管化来增进皮瓣的成活。SVFs在人体脂肪组织中储量巨大、方便获取,自体应用不存在免疫排斥的特性,从脂肪组织新鲜分离得到的SVFs中即含有足够数量的、具有生物学活性的自体ASCs。更重要的是,应用自体SVFs甫助脂肪移植,手术亦可一期完成,且无需体外培养,安全性高,更易于临床推广,这使得脂肪SVFs有望成为促进游离移植脂肪再血管化最有前景的种子细胞。
目前为止,尽管SVFs甫助脂肪移植技术无论在实验室还是临床应用中均被证实可以有效地提高移植物的存活率。然而遗憾的是关于移植后脂肪组织存活的潜在机制尚缺乏令人信服的实验室证据。有几个问题有待于解决：(a) SVFs辅助脂肪移植较单纯脂肪移植相比脂肪细胞在移植过程中形态学发生怎样的动态变化?(b)SVFs辅助脂肪移植的脂肪细胞再生的机制及SVFs中启动移植后早期再血管化进程的最主要因素是什么?(c)SVFs辅助脂肪移植何种最佳细胞浓度才能更加促进移植脂肪的存活率?
围绕这几个科学问题,本实验先是研究了单纯脂肪移植组织学的动态变化趋势,通过比较新鲜分离的自体SVFs辅助脂肪移植及单纯脂肪移植在不同时间点的移植效果,观察各时间点移植物湿重测量、HE染色切片分析,免疫组化、PPARγ荧光原位杂交、Western Blot及ELISA等技术来探讨自体SVFs促进脂肪移植物存活率的脂肪细胞再生及再血管化机制,这对于揭示SVF促进游离脂肪移植存活率的具体机制,推动干细胞疗法在脂肪移植中的应用意义重大,为临床应用提供实验依据。
方法
1、单纯游离脂肪移植的模型制备及脂肪移植物存活的评价。
将0.5ml脂肪颗粒用16号针头,按照随机化原则注射于实验裸鼠腰背区皮下的2个受区,每个时间组共注射6只。分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,观察以下几个指标：(1)移植物大体观；(2)湿重及湿重百分比；(3)HE染色切片；(4)免疫组化染色(S100,CD68)。
2、SVFs辅助游离脂肪移植的模型制备及脂肪移植物存活的评价。
将S、F两组移植物随机注入每只裸鼠背部皮下：S组：将数量为1×106/mlSVFs混悬液与0.3ml脂肪混合；F组将0.3mlDMEM完全培养基与0.3ml的脂肪颗粒均匀混合(阴性对照组)。分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,观察以下几个指标：(1)移植物大体观；(2)湿重及湿重百分比；(3)HE染色切片；(4)免疫组化染色(S100,CD68);(5)统计学分析。
3、SVFs辅助游离脂肪移植再血管化的检测
分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,检测S组及F组以下指标：(1)HE染色切片分析；(2)免疫组化染色(CD31、CD34)及系统软件分析血管密度及趋势；(3)Western Blot检测肝细胞生长因子(HGF);(4)ELISA检测血管内皮生长因子(VEGF)、和碱性成纤维细胞生长因子(bFGF)血管相关因子；(5)统计学分析。
4、SVFs辅助游离脂肪移植的脂肪细胞再生的检测
分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,检测S组及F组以下指标：(1)HE染色切片分析；(2)免疫组化染色(CD68、S100)及系统软件分析新生脂肪细胞及成熟脂肪细胞存活率及趋势；(3)PPARγ荧光原位杂交检测来源于人的脂肪因子的表达及分化情况；(4)统计学分析。
5、SVFs辅助游离脂肪移植的最佳浓度的检测
将A、B、C、D四组移植物随机注入每只裸鼠背部皮下(n=6)：A组将数量约5×105/ml的SVFs混悬液,B组将数量为1×106/mlSVFs混悬液,C组将数量为2×106/mlSVFs混悬液分别与O.3ml脂肪混合；D组将0.3mlDMEM完全培养基与0.3ml的脂肪颗粒均匀混合(阴性对照组)。移植术后3个月取材对移植脂肪组织进行观察,指标有：(1)脂肪组织湿重测定；(2)HE切片染色；(3)Gunderson"点计数”法定量评价移植物纤维坏死及脂肪细胞存活情况；(4)移植物中毛细血管数目；(5)统计学分析。
6.统计学处理
数据以均数±标准差(x±s)表示,使用统计软件SPSS13.0进行数据处理。两组之间比较采用配对t检验：不同时间点的差异采用协方差分析；完全随机设计资料的多样本均数比较采用LSD检验。
结果
1、术后不同时间点单纯脂肪移植物存活率：1天(82.98±3.01)%,4天(81.91±2.931)%,7天(82.27±1.74)%,14天(82.98±1.90)%,30天(48.58±3.13)%,60天(47.16±2.49)%,90天(47.52±1.74)%。移植后的脂肪在两周内湿重变化不显著,14周至30天湿重显著性减少,减少约为第一天的一半,随后湿重减少较为缓慢,90天后湿重保持稳定约有47%组织存活。HE观察：移植早期组织中央部出现坏死形成大量空泡,脂肪细胞间隙出现增宽,间质组织明显长入；第7天移出现了少量新生的脂肪细胞,随后新生的脂肪细胞增多并趋向成熟,移植晚期分化成为成熟脂肪细胞且数量逐渐减少,纤维化明显,部分区域组织结构与正常的脂肪组织基本相同。免疫组化染色S-100蛋白阳性及小细胞CD68阴性均证实新生的小细胞是脂肪细胞。
2、SVFs辅助脂肪移植后湿重百分比：1天(83.33±2.49)%,4天(84.04±2.93)%,7天(84.40±2.58)%,14天(87.23±3.01)%,30天(56.03±2.20)%,60天(54.61±1.74)%,90天(56.38±3.50)%,两组采用配对t检验比较除第1、4、7天与对照组没有显著性差异外(P>0.05),余时间组均显著高于对照组,两组比较差异有统计学意义(P<0.05)。不同时间点SVFs组协方差分析示：不同天数之间比较有统计学差异(P<0.05)。SVFs辅助脂肪移植较对照组而言14天移植物存活率稍有抬高,30天存活率减少约为第一天的27%,90天约有56%组织存活。HE观察：早期SVFs组移植物中央区出现空泡、囊腔样改变较对照组少,间质组织长入明显,SVFs组移植后第7天出现了新生的多房的脂肪细胞且较对照组多,同时伴随成熟程度不一的多种形态的新生脂肪细胞存在,晚期分化为新生的成熟脂肪细胞较对照组多。免疫组化染色：新生细胞S-100蛋白阳性及CD68表达阴性。
3、①HE组织学观察SVFs组于移植后第4天包膜出现新生血管较对照组早(第7天),血管密度较对照组大,新生血管逐渐从移植物包膜向中央长入。②ELISA检测不同时间点血管因子VEGF、bFGF表达量SVFs在移植后早中期第4天、7天、14天表达量均较对照组高,且第7天达到峰值,两组采用配对t检验比较差异有统计学意义(P<0.05),30天后的时间点表达降低,两组没有显著性差异(P>0.05)；不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。③estern Blot检测不同时间点HGF血管化细胞因子：除移植后第30天两组没有显著性差异外,各时间点SVFs表达量均较对照组高,有显著性差异(P<0.05),不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。④免疫组化染色：造血干细胞系的CD34早期表达较少,第7天开始增多,14天-30天达到高峰,随后逐渐减少,SVFs组表达高于对照组,形态上从小的间质样细胞分化为成熟的管腔样血管结构。⑤免疫组化系统软件分析CD31阳性血管的密度：SVFs组第4天密度缓慢增高(对照组为第7天),到30天达到高峰,随后趋于平稳。SVFs血管密度均高于对照组,两组采用配对t检验比较于7、14、30天两组血管密度差异有统计学意义(P<0.05),余时间组无差异(P>0.05)。不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。
4、①移植术后HE染色：观察单纯脂肪移植组新生脂肪细胞的动态演变过程：由原始间叶组织衍变成梭形脂肪细胞再演变成前脂肪细胞、圆形脂肪细胞、多泡性脂肪细胞、单泡性印戒样细胞、和成熟脂肪细胞。而SVFs组除了具有对照组特点外在移植早期就以不同分化状态的多种细胞形式出现,且多于对照组。②免疫组化染色系统软件分析S100阳性的成熟脂肪细胞及新生脂肪细胞不同时间点密度变化：新生脂肪细胞移植后7天开始增多,60天达到高峰,随后密度稳定,SVFs组高于对照组；成熟脂肪细胞于移植后密度逐渐减少,14-30天减少到最高值,60天后较为平稳,SVFs组高于对照组。二者采用配对t检验比较分别于14天、30天、60天两组差异有统计学意义(P<0.05),余时间点差异无统计学意义(P>0.05)。不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。③免疫组化染色CD68早期表达较少,晚期逐渐增多,集中在脂肪细胞间隙内,新生脂肪细胞区域表达大部分阴性。④PARy荧光原位杂交证实新生脂肪细胞核及胞浆有点状绿色荧光表达,证实了新生的脂肪细胞大部分是来源于人并具有分化能力,进一步排除了巨噬细胞的肯能。
5.不同浓度组比较：①湿重：A组(60.00±6.32)mg,B组(81.67±7.53)mg, C组(66.70±8.16)mg,D组(45.02±10.49)mg,B组脂肪存活率均高于A、C、D组(P<0.05),A、C两组之间比较差异无统计学意义(P>0.05)。②血管密度：A、C、B组血管密度均高于组D,且B组明显高于其他三组(P<0.05),A、C两组之间比较差异无统计学意义(P>0.05)。③点计数：B组存活脂肪细胞计数均高于A、C、D组(P<0.05),纤维组织计数均低于D组(P<0.05),A、C两组之间比较差异均无统计学意义(P>0.05)
讨论
目前,自体脂肪颗粒移植后的吸收率,国内外的报道尚不一致,脂肪移植后的吸收率从5%-100%相差甚远,多数学者认为,术后1年的吸收率约为50%。一般来说,脂肪体积在移植后的1个月内吸收最快,2-6个月后开始趋于稳定,1年后几乎不再吸收。近几年,人们对脂肪移植的研究更多是关注于临床短期或长期的移植效果及再血管化的效应,尚缺乏一个对移植后脂肪细胞动态变化过程观察的实验研究。本实验研究证实单纯脂肪移植后的脂肪存活率在2周至1个月显著性减少,3个月后基本保持稳定。在移植早期出现了新生的脂肪细胞存在,随着时间推移新生脂肪细胞增多且趋向成熟,最终以成熟脂肪细胞存活下来。
应用自脂肪组织新鲜分离的SVFs复合脂肪颗粒为制备软组织修复材料提供了一种新的方法,它较ASCs而言更具有成活率高、吸收率低、使用安全、制备简便和价格低廉的优点,且不需要长期体外培养,减少了污染,可直接应用于自体移植,能够向成骨、成软骨、成脂肪、成血管和神经等方向分化,且具有明显的促再血管化及提高移植物存活率效应。为了证实SVFs复合脂肪移植与单纯脂肪移植相比在形态学及组织学动态演变过程中的差别,本实验比较了不同时间点两组的大体观及组织学并证实了SVFs辅助脂肪移植比单纯脂肪移植更能提高脂肪移植的存活率,明显降低脂肪的纤维化、液化,早期出现了较单纯脂肪组而言更多的新生多房脂肪细胞,并伴随成熟程度不一的多种形态的新生脂肪细胞存在,揭示了脂肪移植物脂肪细胞再生的潜在机制。
正如众所周知,临床上进行任何移植都必须考虑到血液供应,移植物只有获得了充分的血供才能生存。但早期细胞的营养还主要依靠细胞间液的渗透,但这种渗透也最多能营养到距离毛细血管150μm的脂肪细胞,而超过这个距离的细胞无法获得支持细胞代谢所必须的营养。这也解释了为何单纯脂肪细胞游离移植存活率不高的原因。因此如何能快速重建良好的血运,是脂肪移植成功与否的关键。SVFs所具有的促血管化作用便体现出了极大的应用潜力,但是SVFs通过何种机制来促进游离脂肪移植后血运重建目前仍不清楚。本实验证实了SVFs辅助脂肪移植的再血管化最早发生于移植后的第4天,较单纯脂肪移植要早,血管密度较单纯脂肪组多。SVFs组早期VEGF、HGF和bFGF血管相关因子的表达均较单纯脂肪组高,这说明SVFs在移植的早期处于缺氧状态下,通过旁分泌机制有效的分泌促血管因子,从而加速新生血管从移植物的周围受区以“出芽”方式形成,提高了移植物的存活率。
自体脂肪移植的最大挑战在于维持其长久的效果,因此对于脂肪移植后脂肪细胞到底是以何种方式存活下来的探讨成为研究的热点,关于单纯游离移植脂肪的存活机制,存在两种学术观点：一种是宿主取代论,即宿主的巨噬细胞吞噬坏死脂肪细胞释放的脂滴后,变为成熟脂肪细胞,取代原有的移植细胞；一种为细胞存活论,即宿主的巨噬细胞只是清除部分坏死脂肪细胞释放的脂滴,并不能形成脂肪细胞取代移植的脂肪组织,部分宿主原有的脂肪细胞度过最初的缺血缺氧期后,长期存活下来。本实验证实了SVFs脂肪移植比单纯脂肪组更能促进脂肪细胞细胞的再生。成熟脂肪细胞及新生的脂肪细胞存活率均高于对照组。单纯脂肪移植新生脂肪细胞动态演变过程是由梭形脂肪细胞再演变成圆形脂肪细胞、单泡性印戒样细胞和成熟脂肪细胞。而SVFs组则是以不同分化状态的细胞存活,新生的脂肪细胞大部分是来源于人SVFs中的间质细胞分化及去分化脂肪细胞的再分化,少部分是由原有的脂肪细胞成脂再分化而来,揭示了脂肪细胞再生的潜在机制。
国外的研究者提出在SVFs辅助脂肪移植中通常采用的SVFs与脂肪颗粒的体积比是1：1,即可取得较为理想的脂肪移植效果,但按1：1体积比进行移植并不客观准确,且胶原消化过程过于繁琐,因此如何客观准确的评估移植比例,对于脂肪移植是至关重要的。有研究称每克皮下脂肪大约可获取0.5×106～2.0×106个有活力的SVFs.在我们的研究中已证实1ml人脂肪组织中可提取出0.9×106～3.0×106个SVFs,故单位体积的待移植脂肪可混合更多数量的SVFs。我们认为SVFs的浓度是影响移植效果的重要原因之一,利用浓度比进行移植更为精确和客观,而今关于SVFs辅助脂肪移植的有效浓度尚缺乏准确性的报道,如果加入血管基质成分过多,会使得术后脂肪组织的体积无法预测；反之如果加入血管基质成分太少,移植脂肪组织的体积达不到预期效果。故本实验中研究促进脂肪移植的最佳SVFs浓度,发现1×106/ml的SVFs浓度更能促进移植脂肪组织存活率及新生血管生成,为临床软组织缺损修复提供了实验依据。
结论
1、从单纯脂肪及SVFs辅助脂肪移植后到脂肪成活的整个过程中存在着脂肪细胞动态变化的规律,移植物体积的变化伴随着组织学的变化。
2、SVFs辅助脂肪移植比单纯脂肪移植更能提高脂肪移植再血管化及脂肪细胞再生效果,更能提高移植物的存活率。
3、SVFs辅助脂肪移植的再血管化发生较单纯脂肪移植早,自体SVFs通过旁分泌机制有效的分泌促血管因子以提高脂肪移植物再血管化。
4、SVFs甫助脂肪移植早期出现脂肪细胞再生,并伴随分化成熟程度不一的多种形态的新生脂肪细胞存在,最终以成熟的脂肪细胞而存活下来,揭示了细胞存活论及去分化-再分化机制是SVFs辅助脂肪移植物脂肪细胞再生的潜在机制。
5、1×106/ml数量级的SVFs移植脂肪的存活率最高,更适合用于脂肪移植,具有广阔的临床应用前景。
Background and Objection:
Soft tissue defect caused by trauma,disorder, aging and congenital malformations has been the common problems of plastic surgery.Collagen injections, dermal grafts and synthetic materials implanted in soft tissue augmentation are widely used in clinic. In1889, Meulen reported of autologous transplantation of adipose tissue. It has been over a hundred years of history. The use of body fat transplantation has major advantages:(1) low immunorejection;(2) low trauma;(3) easy to operate. Since the1930s, this technology has been widely used in plastic surgery.
Although the significantly results of fat transplantation, but the long-term outcome is difficult to obtain. High absorption and low survival rate, many complications, limited its clinical application. Free fat graft has the absorption of fat liquefaction, graft, fibrous tissue replacement. It makes the graft ultimately save over time is only40-60%of the initial volume. The study found that fat graft was lack of effective blood supply due to acute ischemia and hypoxia in early transplantation. Before the graft with host to establish an adequate blood supply, adipose tissue can only rely on infiltration and infiltration of surrounding tissue fluid to maintain the nutrient supply. At that time, the central part of the adipose tissue has been ischemia and hypoxia, the occurrence of necrosis, liquefaction. Thus, early adequate blood supply and revascularization is the key to their survival after autologous granular fat transplantation. Therefore, how to enhance graft revascularization in a short time in order to improve the survival rate of transplanted fat is the focus field of present research.
Recently, stem cell-based transplantation therapy has the potential to revolutionize the ability to regenerate damaged fat tissues. A promising stem cell-based transfer appears to bear to effectively promote the revascularization of the transplanted tissue.
The previous studies have shown that adipose tissue-derived stem cells (adipose derived stem cells, ASCs) can promote the revascularization of the rat hind limb ischemia model, ischemic muscle tissue containing the vascular endothelial cells transformed from ASCs. The preliminary study found that human ASCs co-culture with fat particle transplanted into nude mice, can promote angiogenesis. It can significantly improve the survival of fat grafts. However, clinical application of autologous ASCs has its limitations. In vitro proliferation to obtain autologous ASCs apparently is unable to support the clinical applications. In common, it takes at least two to three weeks. It means that patients need to receive two surgeries. Moreover, cultured ASCs are complex, easily contaminated, low-security facilities and equipment demanding, higher cost.
Since ASCs derived from fat in the vascular stroma cells (stromal vascular fraction is cells, SVFs), and SVFs can be isolated. Whether SVF can replace of ASCs as seed cells for the promotion early vascular of free transplanted fat? In2008, Kotaro Yoshimura reported the clinical application of SVFs with free fat transplantation. The study indicated that SVFs with fat transplantation can significantly reduce the fibrosis of fat, improve fat survival of the transplanted cells compared with the pure fat transplantation. Di jk found that SVFs intravenous injection into myocardial infarction mice can significantly promote revascularization of ischemic myocardial tissue. Another study showed that SVFs can improve the flaps survived. SVFs in human adipose tissue reserves are huge, easy isolate to the application of autologous immune rejection characteristics, ie, containing a sufficient number of adipose tissue freshly isolated SVFs. SVFs auxiliary of autologous fat transplantation, surgery can also be a complete, and without in vitro culture, safe, easier to clinic which makes SVFs is the promising cells to promote free transplanted fat revascularization seed cells.
Although SVFs with fat grafts can effectively improve graft survival, it is still lack of studies on the potential mechanisms of survival after transplantation of adipose tissue. There are several questions to be future investigation:(a) morphology of SVFs with fat grafts during transfer (b) regeneration mechanism of SVF with fat grafts (c) SVFs with fat grafts what the best cell concentration can be more to promote the survival of the transplanted fat?
At present study, we observe that histology of pure fat transplant. At each time point, transplantation things wet weight measurement, and analysis of HE staining, immunohistochemistry, Western blot and ELISA tests to investigate the fat cells of fat graft survival of autologous SVFs promote regeneration and revascularization mechanism, which would provide data for clinical application.
Methods and materials
1、Models for simply isolated fat grafts and evaluation of fat grafts survival.
Each of nude mice were injected subcutaneously into the two random points on the back with0.2ml of fat particles using16gauge needle. Four indexes are respectively observed on the lth day,4th day,7th day,14th day,30th day,60th day and90th day post transplantation:(1) General views of fat grafts;(2) Wet weight of fat grafts and percentage of wet weight;(3) Haematoxylin staining.(4) Immunohistochemical staining (S100, CD68)
2、Models for isolated fat grafts through SVFs and evaluation of fat grafts survival.
Fat grafts of two groups are randomly injected subcutaneously into the back of each nude mice:suspension liquid of SVFs(1×106/ml) mixed with0.3ml of fat particles (Group S);0.3ml of DMEM mixed with0.3ml of fat particles(Group F, as a control). Five indexes are respectively observed on the lth day,4th day,7th day,14th day,30th day,60th day and90th day post transplantation:(1) General views of fat grafts;(2) Wet weight of fat grafts and percentage of wet weight;(3) Haematoxylin staining.(4) Immunohistochemical staining (S100, CD68).(5) Statistical analysis.
3、Detection of revascularization in isolated fat grafts through SVFs.
Examinations of both Group S and Group F are carried out in each time point mentioned above:(1) Haematoxylin staining;(2) Immunohistochemical staining (CD31, CD34)and analysis of densities and trends of blood vessels by using system software;(3)Detect HGF vascular factor using Western Blot;(4) Detect VEGF and bFGF using ELISA;(5) Statistical analysis.
4、Detection of regeneration of adipocyte in isolated fat grafts through SVFs.
Examinations of both Group S and Group F are carried out in each time point mentioned above:(1) Haematoxylin staining;(2) Immunohistochemical staining (S100, CD68) and analysis of liabilities and trends of naive fat cells and mature adipose cells by using system software;(3)Detect expression and differentiation of human-derived adipokine by using PPARγ Fluorescence in situ hybridization.(4) Statistical analysis.
5、Detect the optimal concentration of SVFs
0.3ml of fat particles are respectively mixed with SVFs of different concentrations:5×105/ml(Group A);1×106/ml(Group B);2×106/ml(Group C),and with0.3ml of DMEM complete medium(Group D,as a control). Four indexes are observed at3months post transplantation:(1)Wet weight of fat tissue;(2) Haematoxylin staining;(3)' Point-counting' technique is applied to assessed fibrous necrosis of grafts and adipokine survival.(4)The numbers of capillary vessels.(n=6)
6、Statistical analysis.
Results were expressed as the mean standard error. SPSS13.0software was used for data analysis. The data was statistically analyzed by using a Two-way ANOVA. LSD test was used for the multiple comparisons of the mean values. Between the two groups were compared using a paired t-test; differences of the different time points using analysis of covariance. A p-level of0.05was considered to represent a statistical significance.
Results:
1、Simply isolated fat grafts survivals at different time points after transplantion:(82.98±3.01) percentage on lth day;(81.91±2.931) percentage on4th day;(82.27±1.74) percentage on7th day;(82.98±1.90) percentage on14th day;(48.58±3.13) percentage on30th day;(47.16±2.49) percentage on60th day;(47.52±1.74) percentage on90th day.NO significant changes of wet weight of the fat grafts appeared within two weeks, but wet weight approximately reduced to half of the first day by14-30days,also subsequently, increasingly reduced slowly,47percentage of the tissues survived on90th day. Haematoxylin staining indicated:large numbers of necrosis vacuoles were shaped in center of early fat grafts and reached a peak by14days,wides of gap among adipocytes increased, regional fibrosis was obvious; a small number of naive fat cells were found by7days, subsequently increasingly increased and tended to mature,these naive fat cells differentiated into mature adipocytes late in transplantation and the number gradually reduced, organizational structures of fat grafts and normal tissues looked basically the same. Both positive effect of S-100protein and negative effect of CD68in naive cells demonstrated that the newborn naive cells were adipoblasts.
2、Percentage of wet weight in isolated fat grafts through SVFs:(82.98±2.95) percentage on1th day;(84.04±2.93) percentage on4th day;(84.40±2.58) percentage on7th day;(87.23±3.01) percentage on14th day;(56.03±2.20) percentage on30th day;(54.61±1.74) percentage on60th day;(56.38±3.50) percentage on90th day. Compared with control group, it presents no significances on1th day,4th day and7th day using Two-sample t-test (P>0.05),but other groups are si gnificantly larger than control group, The difference of multiple comprare between ex perimental groups is significant(P<0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).Compared with control group, survival of fat grafts through SVFs raise d slightly on4th day,reduced by27percentage of the first day on30th day,56percent age of the grafts survived on90th day. Haematoxylin staining indicated that necrosis vacuoles and cyst-like changes appeared in center fat grafts through SVFs in early tim e,but less than control group,the degree of fibrosis was also milder compared with co ntrol group.On7th day,it appeared larger number of naive multilocular fat cells than control group, companied with existence of newborn fat cells of varying degrees of m aturity. It apperred more mature adipocytes in late time. Immunohistochemical stainin g indicated positive effect of S-100protein and negative effect of CD68in naive cells.
3.Haematoxylin staining indicated:neovascularization of higher vascular density appeared on4th day in capsule of the grafts(SVFs Group) compared with control Group (appeared on7th day),also neovascularization gradually extended into center of the grafts; We detected the expressions of VEGF and bFGF at different time points by using ELISA. The levels of both angiogenic factors in SVFs Group were higher than control Group on4th day,7th day and14th day,also they reached a peak on7th day. Two-sample t-test represents the significant difference(P<0.05),30days after transplantation,the levels went down and there was no significant difference(P>0.05). The expressions of HGF in SVFs group were higher than those in the control group except30th day detected by using Western blot,there were significant differences(P<0.05). Immunohistochemical staining indicated:The levels of CD34expressed by hematopoietic cell lines were low,but begun to increase from7th day and reached a peak by30days,then gradually reduced. Small interstitial-like cells differentiated mature luminal-like vascular structures in morphology. The density of blood vessel with positive effect of CD31was analysed through Immunohistochemistry system software.The density gradually raised on4th day in SVFs group(on7th day in control group) and it reached a peak by14-30days,then leveled off.The vascular density in SVFs group was higher than that in control group, Two-sample t-test represents the significant differences on7th day,14th day and30th day(P<0.05) and there was no difference in other groups(P>0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).
4.①HE staining after transplantation:To observe the dynamic evolution of pure fat transplantation group, the mother of neonatal fat cells to become the spindle lipoblasts is followed into preadipocytes, round lipoblasts:from primitive mesenchymal organization Yan, multivesicular lipoblasts, single bubble signet ring-like cells, and mature fat cells. The SVFs group to a variety of different differentiation status of the cells in the form, and more than the control group.②The immunohistochemical staining system software S100positive changes in the density of mature fat cells and neonatal fat cells at different time points:7days after cell transplantation of neonatal fat mother began to increase60days reached a peak, followed by a stable density. The SVFs higher than the control group, independent sample t-test respectively in14days,30days,60days difference between the two groups was statistically significant (P<0.05), the remaining time points were no significant differences (P>0.05). Mature fat cells in the transplant density gradually decreased by14-30days reduced to a maximum value, is relatively stable after60days, the density is slightly increasing trend, SVFs group was higher. Two groups at14days30days60days the difference was statistically significant (P<0.05), the remaining time points were no significant differences (P>0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).③mmunohistochemical staining expression of CD68early less concentrated in the capsule, advanced gradually increased the expression of most of the negative region of new fat cells.④The PPARy fluorescence in situ hybridization confirmed that the nucleus of newborn fat-like a little green fluorescent expression confirmed that most of the nascent fat cells derived from human to further exclude macrophages.
5. Different concentrations of group:①wet weight:group A (60.0±6.325) mg, group B (81.67±7.528) mg, group C (68.33±7.528) mg, group D (48.33±7.528) mg,B group fat survival rate were higher than A, C, D group (P<0.05), A and C, between the two groups comparison was no significant difference (P>0.05).②ensity of blood vessels:A and C and group B blood vessel density higher than in group D and group B was significantly higher than the other three groups (P<0.05), A and C, between the two groups was no significant difference (P>0.05).③point count:survival of group B the fat cell counts were higher than A, C and D group (P <0.05), fibrous tissue counts were lower than group D (P<0.05), and two groups A and C, There was no difference betweenstatistically significant (P>0.05).
DISCUSSIONS
At present, the absorption rate after transplantation of autologous fat particles, is from5%to100%after the fat transplantation, the absorption rate of about50%after1year. In general, the volume of fat absorbed the fastest within one month, beginning to stabilize after2to6months, almost no absorb any more after one year. In recent, the clinic results of fat grafting and revascularization were concerned. This study showed that the survival rate of transplanted fat pure fat in2weeks to1month significantly decreased and remained stable after three months. The presence of new fat cells in the transplant early, and mature fat cells increased over time, ultimately survived.
A new approach was developed for the preparation of soft tissue repair material. SVFs with fat particles, it has a high survival rate, low absorption, safe to use, low-cost advantages and reduce pollution. It can be directly used in autologous transplantation. SVFs have the potential of osteogenic, chondrogenic, adipogenic, vascular and nerves differentiation, and promote revascularization, improve graft survival rate. At present study, we evaluate the dynamic evolution of morphological and histological differences between SVFs with fat graft and fat graft alone. The data shows that SVFs can increase the survival rate of fat graft transplantation significantly reduced the fibrosis of the fat liquefaction, the early emergence of a more naive multilocular fat cells in the more pure fat group, and accompanied by varying degrees.
As is well known clinic, any transplant must take into account the blood supply, a sufficient blood supply support the graft survive. However, the early nutrition of cells also rely on the infiltration of the interstitial fluid, but the maximum nutrition distance of this penetration is150μm. More than this distance from the cell can not get essential nutrients to support cell metabolism. Therefore, how to quickly rebuild the blood supply is the key to the success of fat grafting. SVFs have to promote vascularization would reflect a promising potential applications. The mechanism of promote revascularization after free fat transplantation is still need further study. The present study demonstrated for the first four days of revascularization of the earliest of SVFs with fat grafts after transplantation, compared with pure fat grafts to early vascular density than pure fat group. The SVFs group of early vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and basic fibroblast cell growth factor (bFGF) expression of vascular endothelial growth factor than those of pure fat group, which SVFs in the lack of early transplantation oxygen state, the secretion of angiogenic factors through paracrine mechanisms, thereby accelerating neovascularization "budding" from around the graft recipient site formation, improved graft survival.
Autologous fat transplantation, the tough challenge is to maintain its long-term result. At present, there are two academic point of view:(1) replaced on the host, the host macrophage phagocytosis of necrotic fat cells release lipid droplets, into mature fat cells, to replace the transplanted cells.(2) cell survival theory, the host macrophages only clear part of the necrotic fat cells release lipid droplets and the formation of fat cells to replace the transplantation of adipose tissue. The present study demonstrated that the SVFs transplantation more than the simple steatosis group to promote the regeneration of fat cells. Neonatal fat cells in the dynamic evolution of pure fat transplantation is followed by the spindle fat cell into a round fat cell, a single bubble the sexual signet ring-like cells and mature fat cells. SVFs group is different differentiation status of the cell survival, most of new fat cells derived from human SVFs in the interstitial cell differentiation and dedifferentiation of fat cells and then differentiation, a small part is original and some fat cells into fat differentiation from, reveals a potential mechanism of fat cell regeneration.
The previous studies demonstrated that SVFs with fat grafts and fat particles in the volume ratio is1:1, to obtain the ideal result of fat transplantation.1:1volume ratio for transplantation is not accurate. Collagen digestion protocol is too complicated. The assessment of the transplant the proportion of fat transplantation is crucial. The previous study showed that per gram fat can be harvested approximately0.5×106-2.0×106of SVFs. At present study, the data indicated that lml of human adipose tissue can be harvested0.9×106-3.0×106SVFs extracted. It showed that the concentration of SVFs is one important factor for transplantation. Concentration is more accurate than transplant.
However, to date, the effective concentration of SVFs auxiliary fat transplantation is yet to be reported. Therefore, the objective of this study was to investigate that the role of the concentration of SVFs if enhance transplantation of adipose tissue survival and angiogenesis.
Conclusion
1. During the entire process of transferred fat survived, graft volume and histology were accompanied changes.
2. SVF with fat grafts can improve fat graft revascularization, fat cell regeneration, increase graft survival more than only fat grafts
3. SVFs auxiliary fat graft revascularization occurred more pure fat transplantation early autologous SVFs secretion of angiogenic factors through paracrine mechanisms to improve fat graft revascularization.
4. The data showed that cell survival, dedifferentiation and re-differentiation maybe the mechanism of fat graft fat cell regeneration.
5.1×106/ml in magnitude has the highest survival rate of SVFs transplanted fat It is more suitable for fat transplantation, has a promising clinical application.
因外伤、疾病、衰老和先天畸形所造成的软组织缺损一直是整形外科学面临的常见问题。为解决上述问题,胶原注射、真皮移植、人工合成材料植入等软组织填充方法被广泛应用于临床。1889年,Vander Meulen首先报道了脂肪组织的自体移植,至今已有百余年的历史。自体脂肪移植具有的低免疫原性、供区低损伤、操作简便等优点使得这项技术自上世纪30年代起便被广泛应用于整形外科。
尽管脂肪移植效果明显,但长期移植的效应难以控制,其高吸收率、低存活率、多并发症,限制了它在临床中的应用。游离脂肪移植物也面临着脂肪液化、移植物吸收、纤维组织替代等问题,它使得移植物随时间推移的最终保存量仅为最初体积的40-60%。研究发现,在移植早期,脂肪移植物缺乏有效的血液供应而处于急性缺血缺氧状态。在移植物与宿主建立充分的血供之前,脂肪组织只能靠周围组织液的浸润和渗透来维持营养供应,这时中央部分的脂肪组织已经由于缺血缺氧时间过长,发生了坏死、液化。由此可见,在自体颗粒脂肪移植后的转归过程中,移植早期建立及时、充分的血供,移植物及时地再血管化是影响其成活体积的关键。因此,如何促进移植物在短时间内血管化以提高移植脂肪的存活率成为了当前研究的焦点。
近年来随着人们对干细胞研究的不断深入,利用干细胞移植疗法来促进移植物的再血管化,为有效促进移植组织再血管化展示了美好的前景。Makoto等研究证明脂肪组织来源的干细胞(Adipose derived stem cells, ASCs)可促进大鼠后肢局部缺血模型的再血管化,缺血的肌肉组织含有自ASCs转化的血管内皮细胞。本课题组前期的研究发现,体外培养的人ASCs混合脂肪颗粒移植于裸鼠皮下,促进新生血管形成,显著提高脂肪移植物的存活率。然而,临床应用自体培养的ASCs有其局限性,体外培养获取自体ASCs显然无法完全满足临床需要。体外扩增耗时长,至少需要2-3周的时间,这就意味者接受脂肪移植的患者需要二期手术。不仅如此,体外培养操作繁琐,易污染,安全性低,对设施及器材要求严格,费用较高,临床应用受限制。
既然ASCs来源于脂肪中血管基质层细胞(stromal vascular fraction cells, SVFs),且SVFs可通过新鲜分离获得,那SVFs是否能替代ASCs成为促进游离移植脂肪早期血管化的种子细胞呢?2008年,日本学者Kotaro Yoshimura第一个报道了脂肪中SVFs辅助游离脂肪移植的临床应用,研究证实与单纯脂肪移植相比,SVFs辅助的脂肪移植能明显降低脂肪的纤维化、液化,能显著提高脂肪移植的细胞存活率。A. van Dijk等人发现将SVFs静脉注射入心肌梗死小鼠体内,可显著促进缺血心肌组织的再血管化。最近还有研究指出,SVFs可通过促进小鼠游离皮瓣再血管化来增进皮瓣的成活。SVFs在人体脂肪组织中储量巨大、方便获取,自体应用不存在免疫排斥的特性,从脂肪组织新鲜分离得到的SVFs中即含有足够数量的、具有生物学活性的自体ASCs。更重要的是,应用自体SVFs甫助脂肪移植,手术亦可一期完成,且无需体外培养,安全性高,更易于临床推广,这使得脂肪SVFs有望成为促进游离移植脂肪再血管化最有前景的种子细胞。
目前为止,尽管SVFs甫助脂肪移植技术无论在实验室还是临床应用中均被证实可以有效地提高移植物的存活率。然而遗憾的是关于移植后脂肪组织存活的潜在机制尚缺乏令人信服的实验室证据。有几个问题有待于解决：(a) SVFs辅助脂肪移植较单纯脂肪移植相比脂肪细胞在移植过程中形态学发生怎样的动态变化?(b)SVFs辅助脂肪移植的脂肪细胞再生的机制及SVFs中启动移植后早期再血管化进程的最主要因素是什么?(c)SVFs辅助脂肪移植何种最佳细胞浓度才能更加促进移植脂肪的存活率?
围绕这几个科学问题,本实验先是研究了单纯脂肪移植组织学的动态变化趋势,通过比较新鲜分离的自体SVFs辅助脂肪移植及单纯脂肪移植在不同时间点的移植效果,观察各时间点移植物湿重测量、HE染色切片分析,免疫组化、PPARγ荧光原位杂交、Western Blot及ELISA等技术来探讨自体SVFs促进脂肪移植物存活率的脂肪细胞再生及再血管化机制,这对于揭示SVF促进游离脂肪移植存活率的具体机制,推动干细胞疗法在脂肪移植中的应用意义重大,为临床应用提供实验依据。
方法
1、单纯游离脂肪移植的模型制备及脂肪移植物存活的评价。
将0.5ml脂肪颗粒用16号针头,按照随机化原则注射于实验裸鼠腰背区皮下的2个受区,每个时间组共注射6只。分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,观察以下几个指标：(1)移植物大体观；(2)湿重及湿重百分比；(3)HE染色切片；(4)免疫组化染色(S100,CD68)。
2、SVFs辅助游离脂肪移植的模型制备及脂肪移植物存活的评价。
将S、F两组移植物随机注入每只裸鼠背部皮下：S组：将数量为1×106/mlSVFs混悬液与0.3ml脂肪混合；F组将0.3mlDMEM完全培养基与0.3ml的脂肪颗粒均匀混合(阴性对照组)。分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,观察以下几个指标：(1)移植物大体观；(2)湿重及湿重百分比；(3)HE染色切片；(4)免疫组化染色(S100,CD68);(5)统计学分析。
3、SVFs辅助游离脂肪移植再血管化的检测
分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,检测S组及F组以下指标：(1)HE染色切片分析；(2)免疫组化染色(CD31、CD34)及系统软件分析血管密度及趋势；(3)Western Blot检测肝细胞生长因子(HGF);(4)ELISA检测血管内皮生长因子(VEGF)、和碱性成纤维细胞生长因子(bFGF)血管相关因子；(5)统计学分析。
4、SVFs辅助游离脂肪移植的脂肪细胞再生的检测
分别于移植术后1天、4天、7天、14天、30天、60天、90天共7个时间点,检测S组及F组以下指标：(1)HE染色切片分析；(2)免疫组化染色(CD68、S100)及系统软件分析新生脂肪细胞及成熟脂肪细胞存活率及趋势；(3)PPARγ荧光原位杂交检测来源于人的脂肪因子的表达及分化情况；(4)统计学分析。
5、SVFs辅助游离脂肪移植的最佳浓度的检测
将A、B、C、D四组移植物随机注入每只裸鼠背部皮下(n=6)：A组将数量约5×105/ml的SVFs混悬液,B组将数量为1×106/mlSVFs混悬液,C组将数量为2×106/mlSVFs混悬液分别与O.3ml脂肪混合；D组将0.3mlDMEM完全培养基与0.3ml的脂肪颗粒均匀混合(阴性对照组)。移植术后3个月取材对移植脂肪组织进行观察,指标有：(1)脂肪组织湿重测定；(2)HE切片染色；(3)Gunderson"点计数”法定量评价移植物纤维坏死及脂肪细胞存活情况；(4)移植物中毛细血管数目；(5)统计学分析。
6.统计学处理
数据以均数±标准差(x±s)表示,使用统计软件SPSS13.0进行数据处理。两组之间比较采用配对t检验：不同时间点的差异采用协方差分析；完全随机设计资料的多样本均数比较采用LSD检验。
结果
1、术后不同时间点单纯脂肪移植物存活率：1天(82.98±3.01)%,4天(81.91±2.931)%,7天(82.27±1.74)%,14天(82.98±1.90)%,30天(48.58±3.13)%,60天(47.16±2.49)%,90天(47.52±1.74)%。移植后的脂肪在两周内湿重变化不显著,14周至30天湿重显著性减少,减少约为第一天的一半,随后湿重减少较为缓慢,90天后湿重保持稳定约有47%组织存活。HE观察：移植早期组织中央部出现坏死形成大量空泡,脂肪细胞间隙出现增宽,间质组织明显长入；第7天移出现了少量新生的脂肪细胞,随后新生的脂肪细胞增多并趋向成熟,移植晚期分化成为成熟脂肪细胞且数量逐渐减少,纤维化明显,部分区域组织结构与正常的脂肪组织基本相同。免疫组化染色S-100蛋白阳性及小细胞CD68阴性均证实新生的小细胞是脂肪细胞。
2、SVFs辅助脂肪移植后湿重百分比：1天(83.33±2.49)%,4天(84.04±2.93)%,7天(84.40±2.58)%,14天(87.23±3.01)%,30天(56.03±2.20)%,60天(54.61±1.74)%,90天(56.38±3.50)%,两组采用配对t检验比较除第1、4、7天与对照组没有显著性差异外(P>0.05),余时间组均显著高于对照组,两组比较差异有统计学意义(P<0.05)。不同时间点SVFs组协方差分析示：不同天数之间比较有统计学差异(P<0.05)。SVFs辅助脂肪移植较对照组而言14天移植物存活率稍有抬高,30天存活率减少约为第一天的27%,90天约有56%组织存活。HE观察：早期SVFs组移植物中央区出现空泡、囊腔样改变较对照组少,间质组织长入明显,SVFs组移植后第7天出现了新生的多房的脂肪细胞且较对照组多,同时伴随成熟程度不一的多种形态的新生脂肪细胞存在,晚期分化为新生的成熟脂肪细胞较对照组多。免疫组化染色：新生细胞S-100蛋白阳性及CD68表达阴性。
3、①HE组织学观察SVFs组于移植后第4天包膜出现新生血管较对照组早(第7天),血管密度较对照组大,新生血管逐渐从移植物包膜向中央长入。②ELISA检测不同时间点血管因子VEGF、bFGF表达量SVFs在移植后早中期第4天、7天、14天表达量均较对照组高,且第7天达到峰值,两组采用配对t检验比较差异有统计学意义(P<0.05),30天后的时间点表达降低,两组没有显著性差异(P>0.05)；不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。③estern Blot检测不同时间点HGF血管化细胞因子：除移植后第30天两组没有显著性差异外,各时间点SVFs表达量均较对照组高,有显著性差异(P<0.05),不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。④免疫组化染色：造血干细胞系的CD34早期表达较少,第7天开始增多,14天-30天达到高峰,随后逐渐减少,SVFs组表达高于对照组,形态上从小的间质样细胞分化为成熟的管腔样血管结构。⑤免疫组化系统软件分析CD31阳性血管的密度：SVFs组第4天密度缓慢增高(对照组为第7天),到30天达到高峰,随后趋于平稳。SVFs血管密度均高于对照组,两组采用配对t检验比较于7、14、30天两组血管密度差异有统计学意义(P<0.05),余时间组无差异(P>0.05)。不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。
4、①移植术后HE染色：观察单纯脂肪移植组新生脂肪细胞的动态演变过程：由原始间叶组织衍变成梭形脂肪细胞再演变成前脂肪细胞、圆形脂肪细胞、多泡性脂肪细胞、单泡性印戒样细胞、和成熟脂肪细胞。而SVFs组除了具有对照组特点外在移植早期就以不同分化状态的多种细胞形式出现,且多于对照组。②免疫组化染色系统软件分析S100阳性的成熟脂肪细胞及新生脂肪细胞不同时间点密度变化：新生脂肪细胞移植后7天开始增多,60天达到高峰,随后密度稳定,SVFs组高于对照组；成熟脂肪细胞于移植后密度逐渐减少,14-30天减少到最高值,60天后较为平稳,SVFs组高于对照组。二者采用配对t检验比较分别于14天、30天、60天两组差异有统计学意义(P<0.05),余时间点差异无统计学意义(P>0.05)。不同时间点SVFs组协方差分析示天数之间比较有统计学差异(P<0.05)。③免疫组化染色CD68早期表达较少,晚期逐渐增多,集中在脂肪细胞间隙内,新生脂肪细胞区域表达大部分阴性。④PARy荧光原位杂交证实新生脂肪细胞核及胞浆有点状绿色荧光表达,证实了新生的脂肪细胞大部分是来源于人并具有分化能力,进一步排除了巨噬细胞的肯能。
5.不同浓度组比较：①湿重：A组(60.00±6.32)mg,B组(81.67±7.53)mg, C组(66.70±8.16)mg,D组(45.02±10.49)mg,B组脂肪存活率均高于A、C、D组(P<0.05),A、C两组之间比较差异无统计学意义(P>0.05)。②血管密度：A、C、B组血管密度均高于组D,且B组明显高于其他三组(P<0.05),A、C两组之间比较差异无统计学意义(P>0.05)。③点计数：B组存活脂肪细胞计数均高于A、C、D组(P<0.05),纤维组织计数均低于D组(P<0.05),A、C两组之间比较差异均无统计学意义(P>0.05)
讨论
目前,自体脂肪颗粒移植后的吸收率,国内外的报道尚不一致,脂肪移植后的吸收率从5%-100%相差甚远,多数学者认为,术后1年的吸收率约为50%。一般来说,脂肪体积在移植后的1个月内吸收最快,2-6个月后开始趋于稳定,1年后几乎不再吸收。近几年,人们对脂肪移植的研究更多是关注于临床短期或长期的移植效果及再血管化的效应,尚缺乏一个对移植后脂肪细胞动态变化过程观察的实验研究。本实验研究证实单纯脂肪移植后的脂肪存活率在2周至1个月显著性减少,3个月后基本保持稳定。在移植早期出现了新生的脂肪细胞存在,随着时间推移新生脂肪细胞增多且趋向成熟,最终以成熟脂肪细胞存活下来。
应用自脂肪组织新鲜分离的SVFs复合脂肪颗粒为制备软组织修复材料提供了一种新的方法,它较ASCs而言更具有成活率高、吸收率低、使用安全、制备简便和价格低廉的优点,且不需要长期体外培养,减少了污染,可直接应用于自体移植,能够向成骨、成软骨、成脂肪、成血管和神经等方向分化,且具有明显的促再血管化及提高移植物存活率效应。为了证实SVFs复合脂肪移植与单纯脂肪移植相比在形态学及组织学动态演变过程中的差别,本实验比较了不同时间点两组的大体观及组织学并证实了SVFs辅助脂肪移植比单纯脂肪移植更能提高脂肪移植的存活率,明显降低脂肪的纤维化、液化,早期出现了较单纯脂肪组而言更多的新生多房脂肪细胞,并伴随成熟程度不一的多种形态的新生脂肪细胞存在,揭示了脂肪移植物脂肪细胞再生的潜在机制。
正如众所周知,临床上进行任何移植都必须考虑到血液供应,移植物只有获得了充分的血供才能生存。但早期细胞的营养还主要依靠细胞间液的渗透,但这种渗透也最多能营养到距离毛细血管150μm的脂肪细胞,而超过这个距离的细胞无法获得支持细胞代谢所必须的营养。这也解释了为何单纯脂肪细胞游离移植存活率不高的原因。因此如何能快速重建良好的血运,是脂肪移植成功与否的关键。SVFs所具有的促血管化作用便体现出了极大的应用潜力,但是SVFs通过何种机制来促进游离脂肪移植后血运重建目前仍不清楚。本实验证实了SVFs辅助脂肪移植的再血管化最早发生于移植后的第4天,较单纯脂肪移植要早,血管密度较单纯脂肪组多。SVFs组早期VEGF、HGF和bFGF血管相关因子的表达均较单纯脂肪组高,这说明SVFs在移植的早期处于缺氧状态下,通过旁分泌机制有效的分泌促血管因子,从而加速新生血管从移植物的周围受区以“出芽”方式形成,提高了移植物的存活率。
自体脂肪移植的最大挑战在于维持其长久的效果,因此对于脂肪移植后脂肪细胞到底是以何种方式存活下来的探讨成为研究的热点,关于单纯游离移植脂肪的存活机制,存在两种学术观点：一种是宿主取代论,即宿主的巨噬细胞吞噬坏死脂肪细胞释放的脂滴后,变为成熟脂肪细胞,取代原有的移植细胞；一种为细胞存活论,即宿主的巨噬细胞只是清除部分坏死脂肪细胞释放的脂滴,并不能形成脂肪细胞取代移植的脂肪组织,部分宿主原有的脂肪细胞度过最初的缺血缺氧期后,长期存活下来。本实验证实了SVFs脂肪移植比单纯脂肪组更能促进脂肪细胞细胞的再生。成熟脂肪细胞及新生的脂肪细胞存活率均高于对照组。单纯脂肪移植新生脂肪细胞动态演变过程是由梭形脂肪细胞再演变成圆形脂肪细胞、单泡性印戒样细胞和成熟脂肪细胞。而SVFs组则是以不同分化状态的细胞存活,新生的脂肪细胞大部分是来源于人SVFs中的间质细胞分化及去分化脂肪细胞的再分化,少部分是由原有的脂肪细胞成脂再分化而来,揭示了脂肪细胞再生的潜在机制。
国外的研究者提出在SVFs辅助脂肪移植中通常采用的SVFs与脂肪颗粒的体积比是1：1,即可取得较为理想的脂肪移植效果,但按1：1体积比进行移植并不客观准确,且胶原消化过程过于繁琐,因此如何客观准确的评估移植比例,对于脂肪移植是至关重要的。有研究称每克皮下脂肪大约可获取0.5×106～2.0×106个有活力的SVFs.在我们的研究中已证实1ml人脂肪组织中可提取出0.9×106～3.0×106个SVFs,故单位体积的待移植脂肪可混合更多数量的SVFs。我们认为SVFs的浓度是影响移植效果的重要原因之一,利用浓度比进行移植更为精确和客观,而今关于SVFs辅助脂肪移植的有效浓度尚缺乏准确性的报道,如果加入血管基质成分过多,会使得术后脂肪组织的体积无法预测；反之如果加入血管基质成分太少,移植脂肪组织的体积达不到预期效果。故本实验中研究促进脂肪移植的最佳SVFs浓度,发现1×106/ml的SVFs浓度更能促进移植脂肪组织存活率及新生血管生成,为临床软组织缺损修复提供了实验依据。
结论
1、从单纯脂肪及SVFs辅助脂肪移植后到脂肪成活的整个过程中存在着脂肪细胞动态变化的规律,移植物体积的变化伴随着组织学的变化。
2、SVFs辅助脂肪移植比单纯脂肪移植更能提高脂肪移植再血管化及脂肪细胞再生效果,更能提高移植物的存活率。
3、SVFs辅助脂肪移植的再血管化发生较单纯脂肪移植早,自体SVFs通过旁分泌机制有效的分泌促血管因子以提高脂肪移植物再血管化。
4、SVFs甫助脂肪移植早期出现脂肪细胞再生,并伴随分化成熟程度不一的多种形态的新生脂肪细胞存在,最终以成熟的脂肪细胞而存活下来,揭示了细胞存活论及去分化-再分化机制是SVFs辅助脂肪移植物脂肪细胞再生的潜在机制。
5、1×106/ml数量级的SVFs移植脂肪的存活率最高,更适合用于脂肪移植,具有广阔的临床应用前景。
Background and Objection:
Soft tissue defect caused by trauma,disorder, aging and congenital malformations has been the common problems of plastic surgery.Collagen injections, dermal grafts and synthetic materials implanted in soft tissue augmentation are widely used in clinic. In1889, Meulen reported of autologous transplantation of adipose tissue. It has been over a hundred years of history. The use of body fat transplantation has major advantages:(1) low immunorejection;(2) low trauma;(3) easy to operate. Since the1930s, this technology has been widely used in plastic surgery.
Although the significantly results of fat transplantation, but the long-term outcome is difficult to obtain. High absorption and low survival rate, many complications, limited its clinical application. Free fat graft has the absorption of fat liquefaction, graft, fibrous tissue replacement. It makes the graft ultimately save over time is only40-60%of the initial volume. The study found that fat graft was lack of effective blood supply due to acute ischemia and hypoxia in early transplantation. Before the graft with host to establish an adequate blood supply, adipose tissue can only rely on infiltration and infiltration of surrounding tissue fluid to maintain the nutrient supply. At that time, the central part of the adipose tissue has been ischemia and hypoxia, the occurrence of necrosis, liquefaction. Thus, early adequate blood supply and revascularization is the key to their survival after autologous granular fat transplantation. Therefore, how to enhance graft revascularization in a short time in order to improve the survival rate of transplanted fat is the focus field of present research.
Recently, stem cell-based transplantation therapy has the potential to revolutionize the ability to regenerate damaged fat tissues. A promising stem cell-based transfer appears to bear to effectively promote the revascularization of the transplanted tissue.
The previous studies have shown that adipose tissue-derived stem cells (adipose derived stem cells, ASCs) can promote the revascularization of the rat hind limb ischemia model, ischemic muscle tissue containing the vascular endothelial cells transformed from ASCs. The preliminary study found that human ASCs co-culture with fat particle transplanted into nude mice, can promote angiogenesis. It can significantly improve the survival of fat grafts. However, clinical application of autologous ASCs has its limitations. In vitro proliferation to obtain autologous ASCs apparently is unable to support the clinical applications. In common, it takes at least two to three weeks. It means that patients need to receive two surgeries. Moreover, cultured ASCs are complex, easily contaminated, low-security facilities and equipment demanding, higher cost.
Since ASCs derived from fat in the vascular stroma cells (stromal vascular fraction is cells, SVFs), and SVFs can be isolated. Whether SVF can replace of ASCs as seed cells for the promotion early vascular of free transplanted fat? In2008, Kotaro Yoshimura reported the clinical application of SVFs with free fat transplantation. The study indicated that SVFs with fat transplantation can significantly reduce the fibrosis of fat, improve fat survival of the transplanted cells compared with the pure fat transplantation. Di jk found that SVFs intravenous injection into myocardial infarction mice can significantly promote revascularization of ischemic myocardial tissue. Another study showed that SVFs can improve the flaps survived. SVFs in human adipose tissue reserves are huge, easy isolate to the application of autologous immune rejection characteristics, ie, containing a sufficient number of adipose tissue freshly isolated SVFs. SVFs auxiliary of autologous fat transplantation, surgery can also be a complete, and without in vitro culture, safe, easier to clinic which makes SVFs is the promising cells to promote free transplanted fat revascularization seed cells.
Although SVFs with fat grafts can effectively improve graft survival, it is still lack of studies on the potential mechanisms of survival after transplantation of adipose tissue. There are several questions to be future investigation:(a) morphology of SVFs with fat grafts during transfer (b) regeneration mechanism of SVF with fat grafts (c) SVFs with fat grafts what the best cell concentration can be more to promote the survival of the transplanted fat?
At present study, we observe that histology of pure fat transplant. At each time point, transplantation things wet weight measurement, and analysis of HE staining, immunohistochemistry, Western blot and ELISA tests to investigate the fat cells of fat graft survival of autologous SVFs promote regeneration and revascularization mechanism, which would provide data for clinical application.
Methods and materials
1、Models for simply isolated fat grafts and evaluation of fat grafts survival.
Each of nude mice were injected subcutaneously into the two random points on the back with0.2ml of fat particles using16gauge needle. Four indexes are respectively observed on the lth day,4th day,7th day,14th day,30th day,60th day and90th day post transplantation:(1) General views of fat grafts;(2) Wet weight of fat grafts and percentage of wet weight;(3) Haematoxylin staining.(4) Immunohistochemical staining (S100, CD68)
2、Models for isolated fat grafts through SVFs and evaluation of fat grafts survival.
Fat grafts of two groups are randomly injected subcutaneously into the back of each nude mice:suspension liquid of SVFs(1×106/ml) mixed with0.3ml of fat particles (Group S);0.3ml of DMEM mixed with0.3ml of fat particles(Group F, as a control). Five indexes are respectively observed on the lth day,4th day,7th day,14th day,30th day,60th day and90th day post transplantation:(1) General views of fat grafts;(2) Wet weight of fat grafts and percentage of wet weight;(3) Haematoxylin staining.(4) Immunohistochemical staining (S100, CD68).(5) Statistical analysis.
3、Detection of revascularization in isolated fat grafts through SVFs.
Examinations of both Group S and Group F are carried out in each time point mentioned above:(1) Haematoxylin staining;(2) Immunohistochemical staining (CD31, CD34)and analysis of densities and trends of blood vessels by using system software;(3)Detect HGF vascular factor using Western Blot;(4) Detect VEGF and bFGF using ELISA;(5) Statistical analysis.
4、Detection of regeneration of adipocyte in isolated fat grafts through SVFs.
Examinations of both Group S and Group F are carried out in each time point mentioned above:(1) Haematoxylin staining;(2) Immunohistochemical staining (S100, CD68) and analysis of liabilities and trends of naive fat cells and mature adipose cells by using system software;(3)Detect expression and differentiation of human-derived adipokine by using PPARγ Fluorescence in situ hybridization.(4) Statistical analysis.
5、Detect the optimal concentration of SVFs
0.3ml of fat particles are respectively mixed with SVFs of different concentrations:5×105/ml(Group A);1×106/ml(Group B);2×106/ml(Group C),and with0.3ml of DMEM complete medium(Group D,as a control). Four indexes are observed at3months post transplantation:(1)Wet weight of fat tissue;(2) Haematoxylin staining;(3)' Point-counting' technique is applied to assessed fibrous necrosis of grafts and adipokine survival.(4)The numbers of capillary vessels.(n=6)
6、Statistical analysis.
Results were expressed as the mean standard error. SPSS13.0software was used for data analysis. The data was statistically analyzed by using a Two-way ANOVA. LSD test was used for the multiple comparisons of the mean values. Between the two groups were compared using a paired t-test; differences of the different time points using analysis of covariance. A p-level of0.05was considered to represent a statistical significance.
Results:
1、Simply isolated fat grafts survivals at different time points after transplantion:(82.98±3.01) percentage on lth day;(81.91±2.931) percentage on4th day;(82.27±1.74) percentage on7th day;(82.98±1.90) percentage on14th day;(48.58±3.13) percentage on30th day;(47.16±2.49) percentage on60th day;(47.52±1.74) percentage on90th day.NO significant changes of wet weight of the fat grafts appeared within two weeks, but wet weight approximately reduced to half of the first day by14-30days,also subsequently, increasingly reduced slowly,47percentage of the tissues survived on90th day. Haematoxylin staining indicated:large numbers of necrosis vacuoles were shaped in center of early fat grafts and reached a peak by14days,wides of gap among adipocytes increased, regional fibrosis was obvious; a small number of naive fat cells were found by7days, subsequently increasingly increased and tended to mature,these naive fat cells differentiated into mature adipocytes late in transplantation and the number gradually reduced, organizational structures of fat grafts and normal tissues looked basically the same. Both positive effect of S-100protein and negative effect of CD68in naive cells demonstrated that the newborn naive cells were adipoblasts.
2、Percentage of wet weight in isolated fat grafts through SVFs:(82.98±2.95) percentage on1th day;(84.04±2.93) percentage on4th day;(84.40±2.58) percentage on7th day;(87.23±3.01) percentage on14th day;(56.03±2.20) percentage on30th day;(54.61±1.74) percentage on60th day;(56.38±3.50) percentage on90th day. Compared with control group, it presents no significances on1th day,4th day and7th day using Two-sample t-test (P>0.05),but other groups are si gnificantly larger than control group, The difference of multiple comprare between ex perimental groups is significant(P<0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).Compared with control group, survival of fat grafts through SVFs raise d slightly on4th day,reduced by27percentage of the first day on30th day,56percent age of the grafts survived on90th day. Haematoxylin staining indicated that necrosis vacuoles and cyst-like changes appeared in center fat grafts through SVFs in early tim e,but less than control group,the degree of fibrosis was also milder compared with co ntrol group.On7th day,it appeared larger number of naive multilocular fat cells than control group, companied with existence of newborn fat cells of varying degrees of m aturity. It apperred more mature adipocytes in late time. Immunohistochemical stainin g indicated positive effect of S-100protein and negative effect of CD68in naive cells.
3.Haematoxylin staining indicated:neovascularization of higher vascular density appeared on4th day in capsule of the grafts(SVFs Group) compared with control Group (appeared on7th day),also neovascularization gradually extended into center of the grafts; We detected the expressions of VEGF and bFGF at different time points by using ELISA. The levels of both angiogenic factors in SVFs Group were higher than control Group on4th day,7th day and14th day,also they reached a peak on7th day. Two-sample t-test represents the significant difference(P<0.05),30days after transplantation,the levels went down and there was no significant difference(P>0.05). The expressions of HGF in SVFs group were higher than those in the control group except30th day detected by using Western blot,there were significant differences(P<0.05). Immunohistochemical staining indicated:The levels of CD34expressed by hematopoietic cell lines were low,but begun to increase from7th day and reached a peak by30days,then gradually reduced. Small interstitial-like cells differentiated mature luminal-like vascular structures in morphology. The density of blood vessel with positive effect of CD31was analysed through Immunohistochemistry system software.The density gradually raised on4th day in SVFs group(on7th day in control group) and it reached a peak by14-30days,then leveled off.The vascular density in SVFs group was higher than that in control group, Two-sample t-test represents the significant differences on7th day,14th day and30th day(P<0.05) and there was no difference in other groups(P>0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).
4.①HE staining after transplantation:To observe the dynamic evolution of pure fat transplantation group, the mother of neonatal fat cells to become the spindle lipoblasts is followed into preadipocytes, round lipoblasts:from primitive mesenchymal organization Yan, multivesicular lipoblasts, single bubble signet ring-like cells, and mature fat cells. The SVFs group to a variety of different differentiation status of the cells in the form, and more than the control group.②The immunohistochemical staining system software S100positive changes in the density of mature fat cells and neonatal fat cells at different time points:7days after cell transplantation of neonatal fat mother began to increase60days reached a peak, followed by a stable density. The SVFs higher than the control group, independent sample t-test respectively in14days,30days,60days difference between the two groups was statistically significant (P<0.05), the remaining time points were no significant differences (P>0.05). Mature fat cells in the transplant density gradually decreased by14-30days reduced to a maximum value, is relatively stable after60days, the density is slightly increasing trend, SVFs group was higher. Two groups at14days30days60days the difference was statistically significant (P<0.05), the remaining time points were no significant differences (P>0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).③mmunohistochemical staining expression of CD68early less concentrated in the capsule, advanced gradually increased the expression of most of the negative region of new fat cells.④The PPARy fluorescence in situ hybridization confirmed that the nucleus of newborn fat-like a little green fluorescent expression confirmed that most of the nascent fat cells derived from human to further exclude macrophages.
5. Different concentrations of group:①wet weight:group A (60.0±6.325) mg, group B (81.67±7.528) mg, group C (68.33±7.528) mg, group D (48.33±7.528) mg,B group fat survival rate were higher than A, C, D group (P<0.05), A and C, between the two groups comparison was no significant difference (P>0.05).②ensity of blood vessels:A and C and group B blood vessel density higher than in group D and group B was significantly higher than the other three groups (P<0.05), A and C, between the two groups was no significant difference (P>0.05).③point count:survival of group B the fat cell counts were higher than A, C and D group (P <0.05), fibrous tissue counts were lower than group D (P<0.05), and two groups A and C, There was no difference betweenstatistically significant (P>0.05).
DISCUSSIONS
At present, the absorption rate after transplantation of autologous fat particles, is from5%to100%after the fat transplantation, the absorption rate of about50%after1year. In general, the volume of fat absorbed the fastest within one month, beginning to stabilize after2to6months, almost no absorb any more after one year. In recent, the clinic results of fat grafting and revascularization were concerned. This study showed that the survival rate of transplanted fat pure fat in2weeks to1month significantly decreased and remained stable after three months. The presence of new fat cells in the transplant early, and mature fat cells increased over time, ultimately survived.
A new approach was developed for the preparation of soft tissue repair material. SVFs with fat particles, it has a high survival rate, low absorption, safe to use, low-cost advantages and reduce pollution. It can be directly used in autologous transplantation. SVFs have the potential of osteogenic, chondrogenic, adipogenic, vascular and nerves differentiation, and promote revascularization, improve graft survival rate. At present study, we evaluate the dynamic evolution of morphological and histological differences between SVFs with fat graft and fat graft alone. The data shows that SVFs can increase the survival rate of fat graft transplantation significantly reduced the fibrosis of the fat liquefaction, the early emergence of a more naive multilocular fat cells in the more pure fat group, and accompanied by varying degrees.
As is well known clinic, any transplant must take into account the blood supply, a sufficient blood supply support the graft survive. However, the early nutrition of cells also rely on the infiltration of the interstitial fluid, but the maximum nutrition distance of this penetration is150μm. More than this distance from the cell can not get essential nutrients to support cell metabolism. Therefore, how to quickly rebuild the blood supply is the key to the success of fat grafting. SVFs have to promote vascularization would reflect a promising potential applications. The mechanism of promote revascularization after free fat transplantation is still need further study. The present study demonstrated for the first four days of revascularization of the earliest of SVFs with fat grafts after transplantation, compared with pure fat grafts to early vascular density than pure fat group. The SVFs group of early vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and basic fibroblast cell growth factor (bFGF) expression of vascular endothelial growth factor than those of pure fat group, which SVFs in the lack of early transplantation oxygen state, the secretion of angiogenic factors through paracrine mechanisms, thereby accelerating neovascularization "budding" from around the graft recipient site formation, improved graft survival.
Autologous fat transplantation, the tough challenge is to maintain its long-term result. At present, there are two academic point of view:(1) replaced on the host, the host macrophage phagocytosis of necrotic fat cells release lipid droplets, into mature fat cells, to replace the transplanted cells.(2) cell survival theory, the host macrophages only clear part of the necrotic fat cells release lipid droplets and the formation of fat cells to replace the transplantation of adipose tissue. The present study demonstrated that the SVFs transplantation more than the simple steatosis group to promote the regeneration of fat cells. Neonatal fat cells in the dynamic evolution of pure fat transplantation is followed by the spindle fat cell into a round fat cell, a single bubble the sexual signet ring-like cells and mature fat cells. SVFs group is different differentiation status of the cell survival, most of new fat cells derived from human SVFs in the interstitial cell differentiation and dedifferentiation of fat cells and then differentiation, a small part is original and some fat cells into fat differentiation from, reveals a potential mechanism of fat cell regeneration.
The previous studies demonstrated that SVFs with fat grafts and fat particles in the volume ratio is1:1, to obtain the ideal result of fat transplantation.1:1volume ratio for transplantation is not accurate. Collagen digestion protocol is too complicated. The assessment of the transplant the proportion of fat transplantation is crucial. The previous study showed that per gram fat can be harvested approximately0.5×106-2.0×106of SVFs. At present study, the data indicated that lml of human adipose tissue can be harvested0.9×106-3.0×106SVFs extracted. It showed that the concentration of SVFs is one important factor for transplantation. Concentration is more accurate than transplant.
However, to date, the effective concentration of SVFs auxiliary fat transplantation is yet to be reported. Therefore, the objective of this study was to investigate that the role of the concentration of SVFs if enhance transplantation of adipose tissue survival and angiogenesis.
Conclusion
1. During the entire process of transferred fat survived, graft volume and histology were accompanied changes.
2. SVF with fat grafts can improve fat graft revascularization, fat cell regeneration, increase graft survival more than only fat grafts
3. SVFs auxiliary fat graft revascularization occurred more pure fat transplantation early autologous SVFs secretion of angiogenic factors through paracrine mechanisms to improve fat graft revascularization.
4. The data showed that cell survival, dedifferentiation and re-differentiation maybe the mechanism of fat graft fat cell regeneration.
5.1×106/ml in magnitude has the highest survival rate of SVFs transplanted fat It is more suitable for fat transplantation, has a promising clinical application.
引文
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