纳米脂肪混合颗粒脂肪移植在瘢痕性面部凹陷及萎缩治疗中的临床作用及相关实验机制

Clinical effect of nano-fat mixed granule fat transplantation in the treatment of cicatricial facial depression and atrophy and the related experimental mechanism

目的了解纳米脂肪混合颗粒脂肪移植在瘢痕性面部凹陷及萎缩治疗中的临床作用,探讨相关实验机制。方法(1)2012年1月—2018年4月,笔者单位收治符合入选标准的瘢痕性面部凹陷及萎缩畸形需行面部脂肪移植患者105例,对其病历资料进行回顾性分析。根据患者意愿,54例10~59岁患者(男12例、女42例)接受传统自体单纯腹部/大腿颗粒脂肪移植,纳入单纯移植组;另51例7~63岁患者(男14例、女37例)接受自体腹部/大腿纳米脂肪混合颗粒脂肪移植,纳入混合移植组。自制量表并根据手术前后照片资料,通过面部饱满度、对称度、瘢痕及并发症等情况评估患者术后3、6个月治疗满意度;术后6个月评估患者是否需行二次手术,计算二次手术率;于患者第2次手术时取其第1次手术移植的脂肪,扫描电子显微镜下观察脂肪细胞形态及微血管情况。(2)从1只4周龄雄性SD大鼠腹部脂肪分离培养脂肪源性干细胞(ADSC),取第5代细胞,培养14d,观察细胞形态,免疫荧光法观察细胞波形蛋白和细胞角蛋白18的表达,行成骨、成脂诱导分化鉴定,流式细胞仪检测CD29、CD44阳性细胞率(样本数为3)。取18只4周龄雄性SD大鼠,按随机数字表法分为ADSC移植组、单纯瘢痕组、空白对照组,每组6只。ADSC移植组、单纯瘢痕组大鼠均经背部皮下注射1mL质量浓度为1mg/mL的溶于磷酸盐缓冲液(PBS)中的博莱霉素溶液建立瘢痕模型,3h后ADSC移植组大鼠于前述注射部位注射1×106个悬于0.1mLPBS中的ADSC,单纯瘢痕组大鼠注射0.1mLPBS;空白对照组大鼠于前述2个时间点在相同部位分别注射相同剂量PBS。各组连续注射28d后,取所有大鼠注射区域的全层皮肤组织,行Masson染色观察胶原纤维情况,免疫组织化学法观察α平滑肌肌动蛋白(α-SMA)和转化生长因子β1(TGF-β1)表达并计数阳性细胞。对数据行Mann-WhitneyU检验、χ2检验、单因素方差分析、LSD检验。结果(1)与术前比较,单纯移植组患者术后3个月面部饱满度、对称度更佳且瘢痕颜色更接近周围皮肤,该组患者术后6个月填充容积较术后3个月有所减少;与术前比较,混合移植组患者术后3、6个月面部饱满度、对称度更佳且瘢痕颜色、质地更接近周围皮肤,该组患者术后6个月填充容积未见较术后3个月明显减少的情况。单纯移植组患者在术后3个月内出现脂肪液化、皮下结节形成各1例。混合移植组患者术后3、6个月治疗满意度均明显高于单纯移植组(Z=-2.566、-3.084,P<0.05或P<0.01)。术后6个月,混合移植组患者二次手术率为7.84%(4/51),明显低于单纯移植组的22.22%(12/54),χ2=4.199,P<0.05。与单纯移植组比较,混合移植组行第2次手术患者第1次手术时受区移植脂肪细胞形态更加饱满、无塌陷干瘪,细胞紧密排列、间隙较小;细胞表面管状、条索状微血管结构更丰富,细胞间隙布满网状分布且长入脂肪组织中的微血管结构。(2)大鼠脂肪分离培养的第5代细胞贴壁生长,呈长梭形或纺锤形,呈鱼群样生长;高表达波形蛋白和细胞角蛋白18;经诱导具有成骨及成脂分化能力;CD29、CD44阳性表达率高于90.00%。细胞鉴定为ADSC。注射28d后,空白对照组大鼠注射区皮肤组织真皮层胶原纤维细密排列;单纯瘢痕组大鼠注射区皮肤组织真皮层大量胶原纤维沉积,纤维束粗大、排列松散不齐,可见大量炎性浸润、大量散在的肌纤维交错分布;ADSC移植组大鼠注射区皮肤组织真皮层中胶原纤维较空白对照组增粗,排列尚整齐,可见少量肌纤维散布、少量炎性浸润。注射28d后,ADSC移植组大鼠注射区皮肤组织中α-SMA主要表达于微血管中,其α-SMA和TGF-β1阳性细胞数分别为每20倍视野下(49±12)、(63±10)个,与空白对照组的每20倍视野下(35±16)、(44±17)个相近(P>0.05),均明显少于单纯瘢痕组的每20倍视野下(135±13)、(121±23)个(P<0.05)。结论与单纯颗粒脂肪移植比较,纳米脂肪混合颗粒脂肪移植治疗瘢痕性面部凹陷及萎缩患者的填充饱满度更好,填充后效果维持更久,瘢痕质地改善更明显。大鼠瘢痕模型实验显示,其机制可能为ADSC抑制α-SMA和TGF-β1的表达,从而抑制瘢痕形成。

Objective To study the clinical effect of nano-fat mixed granule fat transplantation in the treatment of cicatricial facial depression and atrophy, and to explore the related experimental mechanism. Methods (1) From January 2012 to April 2018, 105 patients conforming to the inclusion criteria, with cicatricial facial depression and atrophy deformity who needed facial fat transplantation, were admitted to our unit. Their medical records were analyzed retrospectively. According to the patients′ wishes, 54 patients (12 males and 42 females) aged 10-59 years received traditional transplantation of pure autologous granule fat collected from abdomen/thigh and were included in simple transplantation group, while the other 51 patients (14 males and 37 females) aged 7-63 years received transplantation of autologous nano-fat mixed granule fat collected from abdomen/thigh and were included in mixed transplantation group. The treatment satisfaction of patients 3 and 6 months after operation was assessed by the facial fullness, symmetry, scar, and complications using self-made scales and photo data before and after operation. Six months after operation, the patients were assessed whether they needed to undergo a second operation, and the rate of second operation was calculated. During the second operation, the fat of patients transplanted in the first operation was collected, and the morphology of adipocytes and microangiogenesis was observed under a scanning electron microscope.(2) Adipose-derived stem cells (ADSCs) were isolated and cultured from abdominal fat of a 4-week-old male Sprague-Dawley (SD) rat. The 5th passage of cells were selected to observe cell morphology after cultured for 14 days, observe expression of vimentin and cytokeratin-18 by immunofluorescence method, identify osteogenic and adipogenic differentiation, and detect rates of CD29 and CD44 positive cells by flow cytometer (n=3). Eighteen 4-week-old male SD rats were divided into ADSCs transplantation group, simple scar group, and blank control group according to the random number table, with 6 rats in each group. Rats in ADSCs transplantation group and simple scar group were subcutaneously injected with 1 mL bleomycin which was dissolved in phosphate buffered saline (PBS) with a mass concentration of 1 mg/mL at the back to establish scar models. After 3 hours, rats in ADSCs transplantation group were injected with 1×106 ADSCs suspended in 0.1 mL PBS at the same injection site, while rats in simple scar group were injected with 0.1 mL PBS. Rats in blank control group were injected with the same doses of PBS in the same place at the same two time points mentioned above. After continuous injection for 28 days in each group, the full-thickness skin tissue of the injected area of all rats was collected to observe the collagen fibers by Masson staining and expressions of α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1) by immunohistochemistry, and the positive cells were counted. Data were processed with Mann-Whitney U test,χ2 test, one-way analysis of variance, and least significant difference test. Results (1) Compared with the preoperative condition, the facial fullness and symmetry of patients in simple transplantation group were better in 3 months after operation, with scar color closer to the surrounding skin, and the filling volume of patients in this group decreased in 6 months after operation as compared with that in 3 months after operation. In mixed transplantation group, the facial fullness and symmetry of patients were better in 3 and 6 months after operation as compared with the preoperative condition, with scar color and texture closer to the surrounding skin, and the filling volume in 6 months after operation was not obviously reduced as compared with that in 3 months after operation. Fat liquefaction and subcutaneous nodule formation occurred respectively in 1 patient in simple transplantation group within 3 months after operation. The treatment satisfaction of patients in mixed transplantation group was significantly higher than that in simple transplantation group in 3 and 6 months after operation (Z=-2.566,-3.084, P<0.05 or P<0.01). Six months after operation, the second operation rate of patients in mixed transplantation group was 7.84%(4/51), which was significantly lower than 22.22%(12/54) in simple transplantation group (χ2=4.199, P<0.05). At the second operation, compared with those of simple transplantation group, the cells of fat transplanted in the first operation of patients in mixed transplantation group were more plump, without collapse or dryness, and the cells were closely arranged, with smaller gap;the tubular and the cord-like microvascular structure on the cell surface were more abundant, and the cell gap was full of network-like microvascular structure that grew into the adipose tissue.(2) The fifth passage of cells isolated and cultured from rat fat grew adherently to the wall, with long fusiform or spindle shape, showing shoal-of-fish-like growth. Vimentin and cytokeratin-18 were highly expressed in the cells. Cells showed osteogenic and adipogenic differentiation ability by induction. The positive expression rates of CD29 and CD44 were higher than 90.00%. The cells were identified as ADSCs. After 28 days of injection, the collagen fibers in the dermis of skin tissue at the injection area of rats in blank control group were finely arranged. In simple scar group, a large amount of collagen was deposited in the dermis of skin tissue at the injection area of rats, the fiber bundles were thick and loosely unevenly arranged, and a large number of inflammatory infiltration and scattered muscle fibers were observed. In ADSCs transplantation group, the collagen fibers in the dermis of skin tissue at the injection area of rats were thicker than those of blank control group, with still neat arrangement, and a small amount of scattered muscle fiber and inflammatory infiltration was observed. After 28 days of injection, the expression of α-SMA in ADSCs transplantation group was mainly in microvessels in the dermis of skin tissue at the injected area of rats, and the number of α-SMA and TGF-β1 positive cells was (49±12) and (63±10) cells per 20-fold field of view, respectively, which was similar to (35±16) and (44±17) cells per 20-fold field of view of blank control group (P>0.05), all significantly less than (135±13) and (121±23) cells per 20-fold field of view of simple scar group (P<0.05). Conclusions Compared with those of autologous simple granule fat transplantation, autologous nano-fat mixed granule fat transplantation has better filling fullness in the treatment of patients with scar facial depression and atrophy. The filling effect lasts longer, and the improvement of scar texture is more obvious. As showed in the rat scar model experiment, the mechanism may be that ADSCs inhibit the expressions of α-SMA and TGF-β1, thus inhibiting the formation of scar.