CN113456807A - 一种软组织填充剂及其应用 - Google Patents
一种软组织填充剂及其应用 Download PDFInfo
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- CN113456807A CN113456807A CN202110960706.2A CN202110960706A CN113456807A CN 113456807 A CN113456807 A CN 113456807A CN 202110960706 A CN202110960706 A CN 202110960706A CN 113456807 A CN113456807 A CN 113456807A
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- adipose
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- collagen
- derived stem
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Abstract
本发明公开了一种软组织填充剂,所述软组织填充剂的原料包括:胶原蛋白、脂肪干细胞、细胞生长因子和交联剂。本发明采用胶原蛋白和脂肪干细胞介导移植隆胸,脂肪干细胞能明显提高移植脂肪细胞的成活率,脂肪干细胞分化的组织与胶原蛋白相结合,搭配使用促进细胞生长的细胞生长因子,能够进一步地抑制移植脂肪的不良吸收、感染、坏死,促进移植脂肪组织的血管再生,提高移植脂肪的成活率,有利于乳房的塑形。本发明不会产生异物反应,安全可靠,具有获取方便、来源丰富、填充效果好以及创伤小等优势。
Description
本申请是申请日为2018年08月27日、申请号为201810978453.X、发明名称为《一种基于自体组织的制剂及其应用》的分案申请。
技术领域
本发明涉及生物制剂技术领域,具体涉及一种基于自体组织的制剂及其应用。
背景技术
隆胸又称隆乳或丰胸,是通过植入医用材料或移植自身组织,使乳房体积扩大、形态丰满匀称,改善女性体型、恢复女性特有曲线美的一种手术。从19世纪80年代初,隆胸先后经历了液体石蜡注射隆胸,硅油、硅胶假体乳房植入等,但由于其相关并发症,包括挛缩、变形、变硬、渗漏、手感不自然等问题一直困惑着求医者和医生。因此,寻找安全、有效、无毒、持久的组织代用品成了隆胸发展的关键。
自体脂肪是从人体自身某些部位吸取多余的皮下脂肪细胞,然后经过吸出的混合物经净化处理、注入药物得到复合脂肪颗粒。当人们意识到自体脂肪有可能成为丰乳的材料时,立即受到求医者和施术者的欢迎,并赋以了极大期望。自体脂肪注射隆胸,从19世纪末开始进入临床,由于其手感自然、创口小、技术的不断改进成熟,接受手术治疗者日益增多。然而自体脂肪注射隆胸的成败与否和脂肪细胞的成活率有关,脂肪组织注射后存在吸收等问题,因此常常需要多次手术,并且有液化感染等并发症难题。
鉴于此,提出本发明的内容。
发明内容
为了解决现有技术存在的问题,本发明的目的在于提供一种基于自体组织的制剂及其应用。
为实现上述目的,本发明提供了一种软组织填充剂,所述软组织填充剂的原料包括:胶原蛋白、脂肪干细胞、细胞生长因子和交联剂。
优选的,所述软组织填充剂包括以下重量份的原料:胶原蛋白50~80份、脂肪干细胞20~5份、细胞生长因子10~30份和交联剂1~20份。
优选的,所述软组织填充剂的制备方法包括:将胶原蛋白、脂肪干细胞、细胞生长因子和交联剂混合均匀,置于4℃无菌保存。
优选的,所述胶原蛋白的制备方法包括:
对血液进行分离处理,得到血液的血浆层、白膜层和红细胞层,混合血液的血浆层和白膜层得到混合物,纯化混合物得到胶原蛋白;
所述血浆层和白膜层的体积比为1:1。
优选的,所述脂肪干细胞的制备方法包括:
将脂肪组织与生理盐水混合后进行离心得到纯脂肪组织,酶解脂肪组织得到初代脂肪干细胞,传代培养脂肪干细胞得到第三代脂肪单细胞,待第三代脂肪干细胞的融合率大于等于80%时,得到脂肪干细胞。
优选的,所述细胞生长因子包括成纤维细胞生长因子、表皮生长因子和类胰岛素生长因子中的一种或多种。
优选的,所述交联剂包括1,4-丁二醇缩水甘油醚和聚乙二醇二缩甘油醚中的一种或两种。
优选的,所述软组织填充剂的剂型包括注射剂。
本发明还提供了当制备原料为胶原蛋白和脂肪干细胞的软组织填充剂的制备方法,包括以下步骤:
步骤一、自体胶原蛋白的制备
通过采血针对人体进行采血,血液经首次离心后进入全血分离器,并在全血分离器内进行二次离心,全血离心分为三层,自上而下依次为血浆层、白膜层和红细胞层,将血浆层、白膜层和红细胞层的液体分别通过导管导入不同的储存单元分开存放,由储存单元取出适量血浆层和白膜层液体按比例混合,并经层析单元进行初步纯化、透滤单元进一步纯化即得所需的自体胶原蛋白;
步骤二、自体脂肪干细胞的制备
(1)脂肪干细胞分离:获取位于腹部的自体脂肪组织,加入生理盐水,混合,以转速1200~1800r/min离心10~20min,去除残留的血细胞和组织碎片,获取脂肪组织,向所述脂肪组织按体积比1:2~3加入质量浓度为0.075~0.1%的Ⅰ型胶原酶溶液,于37℃恒温水浴消化30-60min,以转速1000~1500r/min离心2~6min,弃掉上层脂肪组织及下层悬液,获得脂肪干细胞沉淀;
(2)脂肪干细胞培养:将获得的脂肪干细胞沉淀加入DMEM培养基重悬,置于37℃、5%CO2培养,传代,至第3代终止,当所述脂肪间充质干细胞的融合度达80%以上时,收集细胞使用或冻存;
步骤三、制作制剂
将步骤一制得的自体胶原蛋白和步骤二制得的自体脂肪干细胞,混合均匀,置于4℃无菌保存。
优选的,所述制剂还包括促进细胞生长的细胞生长因子,按照重量百分含量计,所述制剂包括自体胶原蛋白50~80%、自体脂肪干细胞20~50%、促进细胞生长的细胞生长因子10~30%。
优选的,所述细胞生长因子包括成纤维细胞生长因子、表皮生长因子、类胰岛素生长因子中的一种或多种。
优选的,所述制剂还包括重量百分含量为1~20%的交联剂。
优选的,所述交联剂选自1,4-丁二醇缩水甘油醚、聚乙二醇二缩甘油醚中的一种或两种。
本发明还提供了上述软组织填充剂在制备填充软组织的药物中的应用。
优选的,所述软组织包括胸部软组织。
乳房由结缔组织和脂肪组织组成,挺拔丰满的乳房很大程度上依靠结缔组织的承托。而胶原蛋白是结缔组织的主要成分,在结缔组织中胶原蛋白常与多糖蛋白相互交织成网状结构,产生一定机械强度,是承托人体曲线,体现挺拔体态的物质基础。脂肪干细胞具有多分化潜能,且遗传稳定性,是组织工程和再生医学理想的种子细胞。
本发明实施例的自体胶原蛋白由人体静脉血提取的富集大量血小板和胶原蛋白的自体胶原蛋白制成,血液经离心分为三层,血浆层主要包含血浆、水、蛋白质、盐类和各种离子等,白膜层主要包括富含血小板区、富含淋巴细胞区、富含单核细胞区和富含粒细胞区,红细胞层可简单分为年轻红细胞和正常红细胞,提取的血浆层和白膜层混合液中含有大量胶原蛋白,同时富含血小板和各种生长因子,进入真皮浅层后,可刺激胶原蛋白、弹性纤维、胶质等的产生,促使组织再生。
脂肪干细胞是从脂肪组织中分离得到的一种具有多向分化潜能的干细胞,研究发现脂肪干细胞能够在体外稳定增殖且衰亡率低,同时它具有取材容易、少量组织即可获取大量干细胞,适宜大规模培养,对机体损伤小等优点,而且其来源广泛,体内储备量大,适宜自体移植的特点。同时在脂肪干细胞在生长增殖过程中能分泌多种细胞生长因子,如成纤维细胞生长因子、角质细胞生长因子等,有促进细胞更新、促进受损细胞组织愈合、增加细胞活力和肌肤免疫力、美容抗衰老等作用。
本发明实施例具有如下优点:
1、本发明实施例采用自体胶原蛋白和自体脂肪干细胞介导的移植隆胸,自体脂肪干细胞能明显提高移植脂肪细胞的成活率,自体脂肪干细胞分化的组织与自体胶原蛋白相结合,进一步搭配使用促进细胞生长的细胞生长因子,能有效地抑制移植脂肪的吸收、感染、坏死,促进移植脂肪组织的血管再生,移植脂肪成活率高、乳房成形效果好。
2、本发明实施例利用自体组织的有效成分进行组织充填,该提取物来自于患者自体组织,不会产生异物反应,安全可靠,具有获取方便、来源丰富、填充效果好以及创伤小等优势。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
本实施例的基于自体组织的注射剂,其制备方法包括:
步骤一、自体胶原蛋白的制备
通过采血针对人体进行采血,血液经首次离心后进入全血分离器,并在全血分离器内进行二次离心,全血离心分为三层,自上而下依次为血浆层、白膜层和红细胞层,将血浆层、白膜层和红细胞层的液体分别通过导管导入不同的储存单元分开存放,由储存单元取出适量血浆层和白膜层液体按1:1混合,并经层析单元进行初步纯化、透滤单元进一步纯化即得所需的自体胶原蛋白;
步骤二、自体脂肪干细胞的制备
(1)脂肪干细胞分离:获取位于腹部的自体脂肪组织,加入生理盐水,混合,以转速1600r/min离心15min,去除残留的血细胞和组织碎片,获取脂肪组织,向所述脂肪组织按体积比1:2加入质量浓度为0.075%的Ⅰ型胶原酶溶液,于37℃恒温水浴消化60min,以转速1200r/min离心4min,弃掉上层脂肪组织及下层悬液,获得脂肪干细胞沉淀;
(2)脂肪干细胞培养:将获得的脂肪干细胞沉淀加入DMEM培养基重悬,置于37℃、5%CO2培养,传代,至第3代终止,当所述脂肪间充质干细胞的融合度达80%以上时,收集细胞使用或冻存;
步骤三、制作制剂
按重量百分含量计,将自体胶原蛋白60%、自体脂肪干细胞25%、成纤维细胞生长因子15%、1,4-丁二醇缩水甘油醚5%,混合均匀,制成制剂,置于4℃无菌保存。
实施例2
一种基于自体组织的注射剂,其制备方法包括:
步骤一、自体胶原蛋白的制备
通过采血针对人体进行采血,血液经首次离心后进入全血分离器,并在全血分离器内进行二次离心,全血离心分为三层,自上而下依次为血浆层、白膜层和红细胞层,将血浆层、白膜层和红细胞层的液体分别通过导管导入不同的储存单元分开存放,由储存单元取出适量血浆层和白膜层液体按1:1混合,并经层析单元进行初步纯化、透滤单元进一步纯化即得所需的自体胶原蛋白;
步骤二、自体脂肪干细胞的制备
(1)脂肪干细胞分离:获取位于腹部的自体脂肪组织,加入生理盐水,混合,以转速1200r/min离心20min,去除残留的血细胞和组织碎片,获取脂肪组织,向所述脂肪组织按体积比1:2加入质量浓度为0.1%的Ⅰ型胶原酶溶液,于37℃恒温水浴消化30min,以转速1500r/min离心2min,弃掉上层脂肪组织及下层悬液,获得脂肪干细胞沉淀;
(2)脂肪干细胞培养:将获得的脂肪干细胞沉淀加入DMEM培养基重悬,置于37℃、5%CO2培养,传代,至第3代终止,当所述脂肪间充质干细胞的融合度达80%以上时,收集细胞使用或冻存;
步骤三、制作制剂
按重量百分含量计,将自体胶原蛋白55%、自体脂肪干细胞20%、表皮生长因子20%、聚乙二醇二缩甘油醚5%,混合均匀,制成制剂,置于4℃无菌保存。
实施例3
一种基于自体组织的注射剂,其制备方法包括:
步骤一、自体胶原蛋白的制备
通过采血针对人体进行采血,血液经首次离心后进入全血分离器,并在全血分离器内进行二次离心,全血离心分为三层,自上而下依次为血浆层、白膜层和红细胞层,将血浆层、白膜层和红细胞层的液体分别通过导管导入不同的储存单元分开存放,由储存单元取出适量血浆层和白膜层液体按1:1混合,并经层析单元进行初步纯化、透滤单元进一步纯化即得所需的自体胶原蛋白;
步骤二、自体脂肪干细胞的制备
(1)脂肪干细胞分离:获取位于腹部的自体脂肪组织,加入生理盐水,混合,以转速1200r/min离心18min,去除残留的血细胞和组织碎片,获取脂肪组织,向所述脂肪组织按体积比1:3加入质量浓度为0.075%的Ⅰ型胶原酶溶液,于37℃恒温水浴消化45min,以转速1200r/min离心6min,弃掉上层脂肪组织及下层悬液,获得脂肪干细胞沉淀;
(2)脂肪干细胞培养:将获得的脂肪干细胞沉淀加入DMEM培养基重悬,置于37℃、5%CO2培养,传代,至第3代终止,当所述脂肪间充质干细胞的融合度达80%以上时,收集细胞使用或冻存;
步骤三、制作制剂
按重量百分含量计,将自体胶原蛋白60%、自体脂肪干细胞20%、表皮生长因子17%、1,4-丁二醇缩水甘油醚3%,混合均匀,制成制剂,置于4℃无菌保存。
实施例4
一种基于自体组织的注射剂,其制备方法包括:
步骤一、自体胶原蛋白的制备
通过采血针对人体进行采血,血液经首次离心后进入全血分离器,并在全血分离器内进行二次离心,全血离心分为三层,自上而下依次为血浆层、白膜层和红细胞层,将血浆层、白膜层和红细胞层的液体分别通过导管导入不同的储存单元分开存放,由储存单元取出适量血浆层和白膜层液体按1:1混合,并经层析单元进行初步纯化、透滤单元进一步纯化即得所需的自体胶原蛋白;
步骤二、自体脂肪干细胞的制备
(1)脂肪干细胞分离:获取位于腹部的自体脂肪组织,加入生理盐水,混合,以转速1200r/min离心20min,去除残留的血细胞和组织碎片,获取脂肪组织,向所述脂肪组织按体积比1:2加入质量浓度为0.09%的Ⅰ型胶原酶溶液,于37℃恒温水浴消化35min,以转速1200r/min离心3min,弃掉上层脂肪组织及下层悬液,获得脂肪干细胞沉淀;
(2)脂肪干细胞培养:将获得的脂肪干细胞沉淀加入DMEM培养基重悬,置于37℃、5%CO2培养,传代,至第3代终止,当所述脂肪间充质干细胞的融合度达80%以上时,收集细胞使用或冻存;
步骤三、制作制剂
按重量百分含量计,将自体胶原蛋白70%、自体脂肪干细胞10%、类胰岛素生长因子12%、聚乙二醇二缩甘油醚8%,混合均匀,制成制剂,置于4℃无菌保存。
实施例5
一种基于自体组织的注射剂,其制备方法包括:
步骤一、自体胶原蛋白的制备
通过采血针对人体进行采血,血液经首次离心后进入全血分离器,并在全血分离器内进行二次离心,全血离心分为三层,自上而下依次为血浆层、白膜层和红细胞层,将血浆层、白膜层和红细胞层的液体分别通过导管导入不同的储存单元分开存放,由储存单元取出适量血浆层和白膜层液体按1:1混合,并经层析单元进行初步纯化、透滤单元进一步纯化即得所需的自体胶原蛋白;
步骤二、自体脂肪干细胞的制备
(1)脂肪干细胞分离:获取位于腹部的自体脂肪组织,加入生理盐水,混合,以转速1500r/min离心20min,去除残留的血细胞和组织碎片,获取脂肪组织,向所述脂肪组织按体积比1:2加入质量浓度为0.1%的Ⅰ型胶原酶溶液,于37℃恒温水浴消化30min,以转速1000r/min离心6min,弃掉上层脂肪组织及下层悬液,获得脂肪干细胞沉淀;
(2)脂肪干细胞培养:将获得的脂肪干细胞沉淀加入DMEM培养基重悬,置于37℃、5%CO2培养,传代,至第3代终止,当所述脂肪间充质干细胞的融合度达80%以上时,收集细胞使用或冻存;
步骤三、制作制剂
按重量百分含量计,将自体胶原蛋白50%、自体脂肪干细胞30%、成纤维细胞生长因子10%、1,4-丁二醇缩水甘油醚10%,混合均匀,制成制剂,置于4℃无菌保存。
测试例
为了说明本发明的由自体组织制成的制剂具有良好的丰胸效果,本发明进行了下列疗效试验。
1、一般资料
选取2015年2月~2017年2月在本机构自愿接受隆胸的60例女性作为研究对象,年龄20~45岁,健康状况良好,无其他系统性疾病,隆胸前均签订知情同意书。纳入标准:①原发性乳房发育不良,胸部扁平。②妊娠后的自发性乳房萎缩。③乳腺组织病变或外伤后的乳房发育不良。④乳房两侧不对称或有轻度下垂者。⑤纳入患者自愿参加实验,并签署知情同意书。排除标准:①心理准备不足者。②乳房组织有炎症或手术切口附近有皮肤炎症者。③患有精神分裂症或精神异常者。④心、肝、肾等重要脏器有病变者。⑤患有免疫系统或造血系统疾病者。⑥乳房癌术后复发或有转移倾向者。将隆胸患者随机分为试验1~5组和对照组,每一组均为10人,两组患者在一般资料上比较差异不明显,不具有统计学意义(P>0.05)。
2、治疗方法
试验组:试验组1~5分别使用本发明实施例1~5制得的注射剂。按设计注射点将本发明实施例制得的注射剂按照皮下层、乳腺下层多隧道多点进行注射,注射完后,按摩使脂肪分布均匀。
对照组:按设计注射点将制得的自体脂肪按照皮下层、乳腺下层多隧道多点进行注射,注射完后,按摩使脂肪分布均匀。
3、疗效判定标准
显效:不同体位下位置、大小对称和谐;手感自然、动感逼真;感觉灵敏。
有效:立位下位置、大小对称和谐;卧位时位置、大小对称和谐性稍差;手感自然、动感稍差;感觉灵敏。
无效:各种体位下位置、大小对称性差;手感不自然、动感差;感觉不灵敏。
4、结果
术后随访3~6个月,两者的治疗效果对比结果见表1。
表1
组别 | 显效 | 有效 | 无效 | 总有效率 |
试验组1 | 8 | 1 | 1 | 90% |
试验组2 | 7 | 2 | 1 | 90% |
试验组3 | 7 | 3 | 0 | 100% |
试验组4 | 8 | 1 | 1 | 90% |
试验组5 | 8 | 2 | 0 | 100% |
对照组 | 2 | 4 | 4 | 60% |
结果表明:试验组1~5的总有效率达到90%以上,明显优于对照组的总有效率60%,差异具有统计学意义。本发明实施例能有效地抑制移植脂肪的吸收、感染、坏死,促进移植脂肪组织的血管再生,移植脂肪成活率高、乳房成形效果好。
虽然,上文中已经用一般性说明及具体实施例对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (9)
1.一种软组织填充剂,其特征在于,所述软组织填充剂的原料包括:胶原蛋白、脂肪干细胞、细胞生长因子和交联剂。
2.根据权利要求1所述的软组织填充剂,其特征在于,所述软组织填充剂包括以下重量份的原料:胶原蛋白50~80份、脂肪干细胞20~5份、细胞生长因子10~30份和交联剂1~20份。
3.根据权利要求1或2所述的软组织填充剂,其特征在于,所述软组织填充剂的制备方法包括:将胶原蛋白、脂肪干细胞、细胞生长因子和交联剂混合均匀,置于4℃无菌保存。
4.根据权利要求1或2所述的软组织填充剂,其特征在于,所述胶原蛋白的制备方法包括:
对血液进行分离处理,得到血液的血浆层、白膜层和红细胞层,混合血液的血浆层和白膜层得到混合物,纯化混合物得到胶原蛋白;
所述血浆层和白膜层的体积比为1:1。
5.根据权利要求1或2所述的软组织填充剂,其特征在于,所述脂肪干细胞的制备方法包括:
将脂肪组织与生理盐水混合后进行离心得到纯脂肪组织,酶解脂肪组织得到初代脂肪干细胞,传代培养脂肪干细胞得到第三代脂肪单细胞,待第三代脂肪干细胞的融合率大于等于80%时,得到脂肪干细胞。
6.根据权利要求1或2所述的软组织填充剂,其特征在于,所述细胞生长因子包括成纤维细胞生长因子、表皮生长因子和类胰岛素生长因子中的一种或多种。
7.根据权利要求1或2所述的软组织填充剂,其特征在于,所述交联剂包括1,4-丁二醇缩水甘油醚和聚乙二醇二缩甘油醚中的一种或两种。
8.权利要求1~7任一项软组织填充剂在制备填充软组织的药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述软组织包括胸部软组织。
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