人脂肪干细胞与部分脱钙骨支架构建组织工程骨的试验研究
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摘要
研究背景
因外伤、感染、肿瘤等原因造成的骨缺损是临床常见疾病,大范围骨缺损的修复目前仍然是临床上难以解决的问题。目前常用自体骨移植、异体骨移植、人工合成骨替代品等方法充填修复骨缺损,这些方法各有其优缺点。自体骨移植需要在患者身上取骨,并发症较多,诸如供区疼痛、失血、感染,供区局部正常结构被破坏、功能受影响,并且自体骨移植来源较少,取骨数量受限。异体和异种骨移植具有免疫原性,可引发机体的排斥反应,并可能导致病原体传播。羟基磷灰石、磷酸钙等常用人工合成骨替代品,可以避免生物源性修复材料的缺陷,但其主要具有充填、支持或骨传导作用,而诱导骨再生能力较弱。生物材料复合BMP等生长因子用于骨缺损修复,由于局部BMP活性、浓度的不确定性以及材料不完全降解的特性,其在体内并不能形成完全的骨组织,达不到理想的骨修复重建作用。因此,如何更好的修复骨组织缺损,一直是修复重建外科领域关注的热点。
近年来,随着材料学、细胞生物学、分子生物学的迅速发展,利用组织工程学原理与方法再生组织甚至器官的特点和优势得到了广泛认同,而骨组织工程研究则是目前公认的该领域最有可能在临床取得实际效益的内容之一。研究表明,组织工程化骨的构建主要涉及三个重要因素,即种子细胞、生物支架、构建方法,其中种子细胞和支架材料是当前研究的重点。成骨细胞在构建组织工程骨方面是比较理想的种子细胞,但是成骨细胞来源不足限制了骨组织工程研究的发展,寻找新的种子细胞来源成为骨组织工程进一步发展的关键内容之一。
自1994年Kaplan从病人皮下脂肪组织中发现异位成骨现象受到启发,提出脂肪前体细胞可以向成骨细胞分化后,至2001年Zuk PA等报道脂肪组织含有的细胞不仅与骨髓间充质干细胞(MSCs)具有相同的干细胞表面标志,也具有向骨、软骨、脂肪、肌肉和神经等细胞分化的能力,明确地提出了脂肪基质中存在具有干细胞特性的细胞——脂肪干细胞(adipose-derivedstem cells,ADSCs)的概念,继之有学者应用脂肪干细胞在免疫功能缺陷的裸鼠皮下形成骨组织。由于脂肪组织在人体分布广泛,尤其是目前整形外科实施的脂肪抽吸术可以提供充足的脂肪组织,平均每300ml脂肪抽吸物可分离出2×10~8～6×10~8个单个核细胞,与MSCs类似,同样来源于胚胎间充质,可以在体外长期培养,有稳定的细胞倍增时间以及较低的细胞老化水平,在原代培养所获得的细胞数量、细胞生长动力学、细胞衰老、多向分化能力以及基因转染效率等方面与MSCs无显著差异,而且两者细胞表型相似,均能特异性表达stro-1蛋白,使脂肪干细胞作为骨组织工程种子细胞的研究成为近年来的研究热点之一。
骨组织工程支架材料是骨组织工程研究的重要内容之一。理想的骨组织工程材料需具有以下特点:良好的生物相容性;良好的生物降解性;适当的力学强度;合适的孔径和孔隙率;具有生物活性,能够介导细胞间信号传导和相互作用,诱导新骨的形成等。目前用于骨组织工程研究的支架材料有二类,一类是天然生物衍生材料,另一类是人工合成材料。脱钙骨(demineralized bone matrix,DBM)来源于骨,经过脱脂、脱钙等处理过程,主要成分为Ⅰ型胶原,具有良好的生物相容性及生物可降解性,并保留了胶原支架的立体多孔状结构,受到研究者们的关注。目前的脱钙骨产品多为异体完全脱钙的骨基质,作为组织工程支架材料时,存在机械强度差、降解快、供受体差异的缺点,供体的性别、年龄、部位以及脱钙骨的制备方法等因素都可能影响脱钙骨的质量。因此,难以满足骨组织工程对支架材料的要求。为克服其不足,部分学者开展了部分脱钙骨作为骨组织工程支架材料的研究。部分脱钙骨(PDBM)是通过对骨组织进行部分脱钙得到的,通过降低脱钙程度,增加脱钙骨的机械强度,在保持脱钙骨的立体多孔状结构的基础上用作骨组织工程支架的一种材料,其与人脂肪干细胞(hADSCs)细胞相容性如何?目前尚未见有报道。
目的
探讨应用人脂肪干细胞作为种子细胞、部分脱钙骨作为支架材料构建组织工程骨的可行性。
方法
1.人脂肪干细胞体外向成骨细胞定向诱导分化的实验研究
(1)从脂肪抽吸物中分离、培养具有多向分化潜能的脂肪干细胞,测定原代(P0)及传代(P1、P2、P5)细胞的生长曲线。
(2)取第二代(P2)细胞为实验组,应用成骨诱导液向成骨细胞定向诱导分化,并以未诱导组为对照组,BM-Purple染色观察碱性磷酸酶(ALP)表达,Von-Kossa染色观察钙结节,免疫荧光检测Ⅰ型胶原、骨钙蛋白(OCN)及骨桥蛋白(OPN),RT-PCR检测Ⅰ型胶原、骨桥蛋白(OPN)及碱性磷酸酶(ALP)mRNA表达。
2.人脂肪干细胞与部分脱钙骨材料细胞相容性实验研究
(1)选用P2细胞用成骨诱导液进行诱导,4d后将细胞与部分脱钙骨材料复合,培养7d后在倒置相差显微镜下观察细胞生长情况;用扫描电镜对细胞材料复合物的表面和横断面进行观察。
(2)用DIO标记细胞后采用激光共聚焦显微镜观察细胞生长情况,并测定细胞在支架材料上的粘附率;ALP定量检测试剂盒检测细胞在材料上表达ALP的情况;放射免疫的方法检测OCN表达。
3.人脂肪干细胞与部分脱钙骨支架体内异位构建组织工程化骨的实验研究
(1)人脂肪干细胞(hADSCs)的分离、培养。
(2)将部分脱钙骨制成10mm×5mm×3mm大小,钴60照射灭菌,将第二代hADSCs按3×10~7/ml接种到部分脱钙骨材料上,将细胞-材料复合物放置于37℃、5%CO2培养箱中孵育4h,待接种细胞已粘附于材料上后,加入10%DMEM诱导培养液在体外培养7d。
(3)裸鼠体内回植:选用5周龄的裸鼠,共16只,分为两组,每组8只,均给与乙醚吸入麻醉,于背部做横切口,长约2cm,向两侧腹部分离皮下间隙,右侧皮下植入hADSCs部分脱钙骨复合物作为实验组,左侧皮下间隙植入无细胞的部分脱钙骨,作为对照组。第一组观察期为8周,第二组观察期为12周。
(4)取材、石蜡切片HE染色,观察成骨情况。
结果:
1.人脂肪干细胞体外向成骨细胞定向诱导分化的实验研究
传代细胞的生长较原代细胞增殖速度快。P_1、P_2、P_5均具有以下共同特征:传代培养的潜伏期约为24 h,传代培养细胞的对数增殖期约为2～3 d,接种后第4～5天进入平台期;细胞诱导14d后,实验组ALP染色成阳性反应,对照组阴性;yon Kossa染色实验组出现深棕色结节状沉积,且随诱导时间增加而加深,对照组没有结节状沉积出现;免疫荧光检测实验组和对照组均表达Ⅰ型胶原,实验组骨钙素(OCN)、骨桥蛋白(OPN)检测为阳性,对照组为阴性或弱阳性;RT—PCR检测表明:实验组诱导14d时有ALP、Osteopontin表达,对照组阴性,实验组、对照组及成骨细胞均有Ⅰ型胶原阳性表达。
2.人脂肪干细胞与部分脱钙骨材料细胞相容性实验研究
扫描电镜观察发现细胞在材料上黏附良好,DIO标记表明细胞在材料上随时间延长逐渐增加,第7天几乎填满所有孔隙。ALP定量检测表明诱导组细胞3d开始检测到ALP表达,14d达到高峰,此后逐渐下降,而非诱导组细ALP始终维持在较低水平。OCN定量检测表明,诱导组和非诱导组随着时间延长,OCN是不断升高的,但诱导组OCN含量明显高于非诱导组。
3.人脂肪干细胞与部分脱钙骨支架裸鼠体内异位构建组织工程化骨的实验研究
第1组(8周时)HE染色,见新生骨小梁呈岛状附着于脱钙骨表面。第2组(12周时)有新骨形成,HE染色,见新生骨小梁呈岛状附着于脱钙骨表面,边缘有一层连续排列的成骨细胞样细胞,有新骨形成。
结论
1.从脂肪抽吸术脂肪抽吸物中可以分离、培养出具有分化潜能的脂肪干细胞,且在体外培养条件下生长良好,第2代细胞在诱导培养下可向成骨细胞分化。
2.人脂肪干细胞与部分脱钙骨材料具有良好的细胞相容性,成骨诱导的人脂肪干细胞能在部分脱钙骨上能较好粘附和生长。细胞在体外三维培养条件下(即支架材料上)仍然表达成骨细胞表型。
3.人脂肪干细胞和部分脱钙骨复合可在裸鼠体内形成组织工程化骨,证实了部分脱钙骨是一种良好的骨组织工程支架材料。
主要创新点
1.对PDBM与成骨诱导的hADSCs的生物相容性进行了研究。
2.对hADSCs在体外以PDBM为支架的三维培养条件下成骨细胞表型ALP、CON进行了定量检测。
3.成功的进行了hADSCs与PDBM支架裸鼠体内异位构建组织工程化骨。
Background
Bone defects caused by trauma,infection,tumor and so on,are one of the common diseases,and the clinic repair of wide-bound bone defects is still hard to tackle at present.Autogenous bones,allogenic/allogenous bones,and artificial bone substitutes are now usually used as obturation materials for bone defects,which have their respective strong and weak points.Surgeries to get autogenous bones cause a lot of complications,such as pains,blood loss,infection,structure and function destroy of donor sites.In addition,the sources of autogenous bones are relatively less,leading to the limited quantity available.Allogenic/allogenous bones are ready to cause reject reaction and may lead to infection of pathogens,due to their immunogenicity.Though artificial bone substitutes,such as hydroxylapatite and calcium phosphate,can make up the shortcomings of biological source-derived repairing materials,they are weak in bone induction,so their applications are limited merely to bone obturation,bone supporting and bone conduction.Biomaterials combined with growth factors,such as BMP,cannot form complete bone tissue in vivo to satisfyingly repair bone defects, due to the indeterminateness of activity and concentration of the regional BMP. Therefore,preferable repair of bone defects has been the focus of reconstructive surgery todate.
Recent development of research on materials,cytobiology,and molecular biology,has made the features and predominances of tissue or organ reconstruction using tissue engineering methods identified with generally.Furthermore,it is presently received that bone tissue engineering is one of the fields which are probable to be put into practice clinically.
Construction of tissue-engineered bone refers to three key factors,namely seed cells,scaffolds,and methods of construction,in which the former two are nowadays the focal points of research.Osteoblasts are relatively ideal seed cells for bone tissue engineering,however,the deficiency of source impedes their further application.So, it is the key point for further development of bone tissue engineering to look for new cell source.
In 1994,Kaplan et al put forward that the precursor cells of adipose could differentiate into osteoblasts as they discovered heterotopic ossification in the subcutaneous fatty tissues of patients.In 2001,Zuk et al reported the proper cells derived from adipose tissue not only expressed the same surface markers as mesenchymal stem cells(MSCs),but also had the potential to differentiate into osteogenesis,chondrogenesis,adipogenesis,myogenesis and neurogenesis, confirming the cells with stem cell features in adipose tissue.Thus,the concept of ADSCs was introduced.Afterwards,subcutaneous ossification by ADSCs in athymic mice which had immunodeficiency was documented.Research on adipose-derived stem cells(ADSCs) as seed cells has become the hot point of tissue engineering recently.Adipose tissue distributes widespreadly in human bodies,and they can be sufficiently provided from liposuction of plastic surgeries.It was reported that 2×10~8～6×10~8 monocytes could be harvested from 300 ml lipoaspirates.ASDCs are similar as MSCs,which are derived from embryo mesenchymal tissues.It was identified by experiments that ADSCs could undergo long-term culture in vitro,have stable doubling time as well as low level of cell senescence,and they are not significantly different from MSCs speaking of harvest rate of primary culture,cell growth kinetics,cell aging,ability of multi-directional differentiation and gene transfection efficiency.Morever,ADSCs and MSCs are similar in phenotype expression,and can both specifically express protein stro-1.
Research on scaffold materials is one of the important fields of bone tissue engineering.Ideal scaffolds for bone tissue engineering have the characteristics such as good biocompatibility,favorable biodegradation,proper mechanical strength, proper aperture and interval porosity,bioactivity,ability to mediate signal transduction and interaction among seeded cells,and potential of osteoinduction.Now there are two kinds of biomaterials usually used for bone tissue engineering,one is natural bio-derivants,the other is artificial synthetic materials.Compared with other biomaterials,demineralized bone matrix(DBM) has distinctive features and so it attracts the attention of the researchers.Bone tissue-derived DBM can be acquired after the processes of defatting and decalcification,which provide it with satisfactory biocompatibility and bio-degradation,due to its essential component of collagen typeⅠand the the remaining stereo-structure with porosity.However,most of the present DBM materials are completely decalcified homologous bone matrix,they are weak in mechanical strength and degrade relatively fast.In addition,the donors' genders,ages, donor sites and the different process methods could probably affect the quality of DBM.Therefore,the traditional DBM materials cannot meet the demands of bone tissue engineering.Partially demineralized bone matrix(PDBM) materials are acquired by partial decalcification of bone tissue.They are used as scaffolds for bone tissue engimeering as they have comparably lower decalcification degree,enhanced mechanical strength as well as stereo-structure with porosity.Researches on compatibility and interaction between PDBM and ADSCs haven't been documented.
Objective:
To investigate the feasibility of tissue-engineered bone construction by PDBM combined with human ADSCs(hADSCs).
Methods
1.In vitro osteoinduction of hADSCs
(1)Human ADSCs were isolated from human lipoasperates,and were further cultured in vitro.Growth curves of passage 0,1,2,5 were tested.The surface markers of Human ADSCs were detected.
(2)Passage 2 hADSCs cultured with osteogenic medium were set as experimental group,uninduced hADSCs were set as control group.Alkaline phosphatase(ALP) of both groups were stained by BM-Purple method,calcium nodes were stained by Von-Kossa method,immunofluorescence observation was performed to detect collagen typeⅠand osteocalcin(OCN) expression,RT-PCR analysis was performed to test collagen typeⅠ,osteopontin(OPN) and ALP mRNA expression.
2.Compatibility assay of PDBM cultured with hADSCs
(1)Passage 2 hADSCs were osteoinduced and seeded onto PDBM in 4 days, inverted phase cntrast microscope was used to observe hADSCs growth after seeding, surface and transaction of the cell/scaffold construction were observed by SEM in 7 days.
(2)The growth of DIO-labeled hADSCs was observed by Laser Scanning Confocal Microscope(LSCM),cell adhesion rate was tested,ALP activity and OCN secretion were also quantitatively measured using specific assay kits.
3,In vivo construction of tissue-engineered bone by PDBM combined with hADSCs
(1)Isolation and in vitro culture of hADSCs.
(2)PDBM materials were cut into cubes with the dimension of 10mm×5mm×3mm.After radiation sterilization by Co 60,passage 2 hADSCs of 3×10~7/ml were seeded onto the cubes.The cell/scaffold constructions were incubated at 37℃with 5%CO2 for 4h.Then osteogenic medium was added and the constructions were subcultured for 7d.
(3)Sixteen 5-week-old athymic mice were averagedly distributed into two groups, After inhalation anesthesia by diethyl ether,2cm-long transverse incisions were performed on the back of the athymic mice,and subcutaneous gaps were disjuncted towards abdomen bilaterally.Then cell/scaffold constructions were implanted subcutaneously at right side as experimental group,PDBM materials without cells were implanted subcutaneously at left side as control group.The observation duration of the first one was 8w,the second one was 12w.
(4)After sacrifice,paraffin sections H&E staining was performed.
Results
1.Experiment of osteogenic differentiation of human adipose-derived stem cells in vitro
Subcultured hADSCs expanded faster than passage 0.Passage 1,2 and 5 hADSCs have features in common:delitescence was about 24h,logistic proliferative phase lasted for 2～3 days,4～5 days after passage the platform phase began;14 days after passage,as for ALP detection,osteoinduced cells stained positive,the controls stained negative;as for Von Kossa staining to observe ossification,osteoinduced cells stained deep brown,demonstrating positive,and the staining became denser with the increase of culture time,however,the controls stained negative;as for immunofluorescence observation,both of the two groups expressed collagen typeⅠpositively,osteoinduced hADSCs expressed OCN and OPN positively,the controls negatively or weak positively;as for RT-PCR analysis,after 14 days of culture, osteoinduced cells expressed ALP and OPN positively,the controls negatively,both of the two groups expressed collagen typeⅠpositively as positive control group.
2.Empirical Study of Biocompatibility of partially Demineralized Bones Matrix Cultured with hADSCs
Human ADSCs adhesived to PDBM well as detected by SEM,DIO-labeled cells went up in number as culture time increased and could almostly stuff all of the pore gaps of PDBM in 7 days.Quantitative assays for ALP and OCN showed that ALP began to express in the induced cells at the 3rd day,7days later the peak value appeared,and ALP content gradually declined since then;however,ALP content in the non-induced cells maintained at a relatively low level;as culture time increased OCN content gradually rised in both induced and non-induced cells.But OCN content in induced group was significant higher than that of non-induced group.Moreover,as culture time increased,the difference was more clear.
3.In vivo construction of tissue-engineered bone by PDBM combined with hADSCs
As H&E staining shown for the forst group,8w after implantation,newly formed bone trabeculas adhesived to the surface of PDBM like islands.Twelve weeks later for the second group,newly formed bone trabeculas also adhesived to the surface of PDBM like islands,along the fringe of which sequentially aligned a layer of osteoblast-like cells.
Conclusion
1.Human ADSCs can be isolated from human lipoasperates,and were further cultured in vitro.Human ADSCs grow fine in vitro,and passage 2 hADSCs can differentiate into osteoblasts cultured with osteogenic medium.
2.Osteoinduced hADSCs adhere to the surface of PDBM well and grow fine on the surface of PDBM,demonstrating that PDBM has good biocompatibility.Human ADSCs can maintain osteoblast phenotype when subjected to osteoinduction under osteo-culture in vitro.
3.Tissue-engineered bone tissue can be formed in vivo by PDBM combined with osteoinduced hADSCs.It is demonstrated that PDBM is a kind of favorable scaffold biomaterials for bone tissue engineering.
Innovation points
1.The biocompatibility of Osteoinduced hADSCs and PDBM were studied.
2.Quantitative assays for ALP and OCN of osteoblast phenotype of Osteoinduced hADSCs were made by three-dimensional scaffold(PDBM) in vitro.
3.It is the first time to constructe tissue-engineered bone by PDBM combined with hADSCs In vivo.
因外伤、感染、肿瘤等原因造成的骨缺损是临床常见疾病,大范围骨缺损的修复目前仍然是临床上难以解决的问题。目前常用自体骨移植、异体骨移植、人工合成骨替代品等方法充填修复骨缺损,这些方法各有其优缺点。自体骨移植需要在患者身上取骨,并发症较多,诸如供区疼痛、失血、感染,供区局部正常结构被破坏、功能受影响,并且自体骨移植来源较少,取骨数量受限。异体和异种骨移植具有免疫原性,可引发机体的排斥反应,并可能导致病原体传播。羟基磷灰石、磷酸钙等常用人工合成骨替代品,可以避免生物源性修复材料的缺陷,但其主要具有充填、支持或骨传导作用,而诱导骨再生能力较弱。生物材料复合BMP等生长因子用于骨缺损修复,由于局部BMP活性、浓度的不确定性以及材料不完全降解的特性,其在体内并不能形成完全的骨组织,达不到理想的骨修复重建作用。因此,如何更好的修复骨组织缺损,一直是修复重建外科领域关注的热点。
近年来,随着材料学、细胞生物学、分子生物学的迅速发展,利用组织工程学原理与方法再生组织甚至器官的特点和优势得到了广泛认同,而骨组织工程研究则是目前公认的该领域最有可能在临床取得实际效益的内容之一。研究表明,组织工程化骨的构建主要涉及三个重要因素,即种子细胞、生物支架、构建方法,其中种子细胞和支架材料是当前研究的重点。成骨细胞在构建组织工程骨方面是比较理想的种子细胞,但是成骨细胞来源不足限制了骨组织工程研究的发展,寻找新的种子细胞来源成为骨组织工程进一步发展的关键内容之一。
自1994年Kaplan从病人皮下脂肪组织中发现异位成骨现象受到启发,提出脂肪前体细胞可以向成骨细胞分化后,至2001年Zuk PA等报道脂肪组织含有的细胞不仅与骨髓间充质干细胞(MSCs)具有相同的干细胞表面标志,也具有向骨、软骨、脂肪、肌肉和神经等细胞分化的能力,明确地提出了脂肪基质中存在具有干细胞特性的细胞——脂肪干细胞(adipose-derivedstem cells,ADSCs)的概念,继之有学者应用脂肪干细胞在免疫功能缺陷的裸鼠皮下形成骨组织。由于脂肪组织在人体分布广泛,尤其是目前整形外科实施的脂肪抽吸术可以提供充足的脂肪组织,平均每300ml脂肪抽吸物可分离出2×10~8～6×10~8个单个核细胞,与MSCs类似,同样来源于胚胎间充质,可以在体外长期培养,有稳定的细胞倍增时间以及较低的细胞老化水平,在原代培养所获得的细胞数量、细胞生长动力学、细胞衰老、多向分化能力以及基因转染效率等方面与MSCs无显著差异,而且两者细胞表型相似,均能特异性表达stro-1蛋白,使脂肪干细胞作为骨组织工程种子细胞的研究成为近年来的研究热点之一。
骨组织工程支架材料是骨组织工程研究的重要内容之一。理想的骨组织工程材料需具有以下特点:良好的生物相容性;良好的生物降解性;适当的力学强度;合适的孔径和孔隙率;具有生物活性,能够介导细胞间信号传导和相互作用,诱导新骨的形成等。目前用于骨组织工程研究的支架材料有二类,一类是天然生物衍生材料,另一类是人工合成材料。脱钙骨(demineralized bone matrix,DBM)来源于骨,经过脱脂、脱钙等处理过程,主要成分为Ⅰ型胶原,具有良好的生物相容性及生物可降解性,并保留了胶原支架的立体多孔状结构,受到研究者们的关注。目前的脱钙骨产品多为异体完全脱钙的骨基质,作为组织工程支架材料时,存在机械强度差、降解快、供受体差异的缺点,供体的性别、年龄、部位以及脱钙骨的制备方法等因素都可能影响脱钙骨的质量。因此,难以满足骨组织工程对支架材料的要求。为克服其不足,部分学者开展了部分脱钙骨作为骨组织工程支架材料的研究。部分脱钙骨(PDBM)是通过对骨组织进行部分脱钙得到的,通过降低脱钙程度,增加脱钙骨的机械强度,在保持脱钙骨的立体多孔状结构的基础上用作骨组织工程支架的一种材料,其与人脂肪干细胞(hADSCs)细胞相容性如何?目前尚未见有报道。
目的
探讨应用人脂肪干细胞作为种子细胞、部分脱钙骨作为支架材料构建组织工程骨的可行性。
方法
1.人脂肪干细胞体外向成骨细胞定向诱导分化的实验研究
(1)从脂肪抽吸物中分离、培养具有多向分化潜能的脂肪干细胞,测定原代(P0)及传代(P1、P2、P5)细胞的生长曲线。
(2)取第二代(P2)细胞为实验组,应用成骨诱导液向成骨细胞定向诱导分化,并以未诱导组为对照组,BM-Purple染色观察碱性磷酸酶(ALP)表达,Von-Kossa染色观察钙结节,免疫荧光检测Ⅰ型胶原、骨钙蛋白(OCN)及骨桥蛋白(OPN),RT-PCR检测Ⅰ型胶原、骨桥蛋白(OPN)及碱性磷酸酶(ALP)mRNA表达。
2.人脂肪干细胞与部分脱钙骨材料细胞相容性实验研究
(1)选用P2细胞用成骨诱导液进行诱导,4d后将细胞与部分脱钙骨材料复合,培养7d后在倒置相差显微镜下观察细胞生长情况;用扫描电镜对细胞材料复合物的表面和横断面进行观察。
(2)用DIO标记细胞后采用激光共聚焦显微镜观察细胞生长情况,并测定细胞在支架材料上的粘附率;ALP定量检测试剂盒检测细胞在材料上表达ALP的情况;放射免疫的方法检测OCN表达。
3.人脂肪干细胞与部分脱钙骨支架体内异位构建组织工程化骨的实验研究
(1)人脂肪干细胞(hADSCs)的分离、培养。
(2)将部分脱钙骨制成10mm×5mm×3mm大小,钴60照射灭菌,将第二代hADSCs按3×10~7/ml接种到部分脱钙骨材料上,将细胞-材料复合物放置于37℃、5%CO2培养箱中孵育4h,待接种细胞已粘附于材料上后,加入10%DMEM诱导培养液在体外培养7d。
(3)裸鼠体内回植:选用5周龄的裸鼠,共16只,分为两组,每组8只,均给与乙醚吸入麻醉,于背部做横切口,长约2cm,向两侧腹部分离皮下间隙,右侧皮下植入hADSCs部分脱钙骨复合物作为实验组,左侧皮下间隙植入无细胞的部分脱钙骨,作为对照组。第一组观察期为8周,第二组观察期为12周。
(4)取材、石蜡切片HE染色,观察成骨情况。
结果:
1.人脂肪干细胞体外向成骨细胞定向诱导分化的实验研究
传代细胞的生长较原代细胞增殖速度快。P_1、P_2、P_5均具有以下共同特征:传代培养的潜伏期约为24 h,传代培养细胞的对数增殖期约为2～3 d,接种后第4～5天进入平台期;细胞诱导14d后,实验组ALP染色成阳性反应,对照组阴性;yon Kossa染色实验组出现深棕色结节状沉积,且随诱导时间增加而加深,对照组没有结节状沉积出现;免疫荧光检测实验组和对照组均表达Ⅰ型胶原,实验组骨钙素(OCN)、骨桥蛋白(OPN)检测为阳性,对照组为阴性或弱阳性;RT—PCR检测表明:实验组诱导14d时有ALP、Osteopontin表达,对照组阴性,实验组、对照组及成骨细胞均有Ⅰ型胶原阳性表达。
2.人脂肪干细胞与部分脱钙骨材料细胞相容性实验研究
扫描电镜观察发现细胞在材料上黏附良好,DIO标记表明细胞在材料上随时间延长逐渐增加,第7天几乎填满所有孔隙。ALP定量检测表明诱导组细胞3d开始检测到ALP表达,14d达到高峰,此后逐渐下降,而非诱导组细ALP始终维持在较低水平。OCN定量检测表明,诱导组和非诱导组随着时间延长,OCN是不断升高的,但诱导组OCN含量明显高于非诱导组。
3.人脂肪干细胞与部分脱钙骨支架裸鼠体内异位构建组织工程化骨的实验研究
第1组(8周时)HE染色,见新生骨小梁呈岛状附着于脱钙骨表面。第2组(12周时)有新骨形成,HE染色,见新生骨小梁呈岛状附着于脱钙骨表面,边缘有一层连续排列的成骨细胞样细胞,有新骨形成。
结论
1.从脂肪抽吸术脂肪抽吸物中可以分离、培养出具有分化潜能的脂肪干细胞,且在体外培养条件下生长良好,第2代细胞在诱导培养下可向成骨细胞分化。
2.人脂肪干细胞与部分脱钙骨材料具有良好的细胞相容性,成骨诱导的人脂肪干细胞能在部分脱钙骨上能较好粘附和生长。细胞在体外三维培养条件下(即支架材料上)仍然表达成骨细胞表型。
3.人脂肪干细胞和部分脱钙骨复合可在裸鼠体内形成组织工程化骨,证实了部分脱钙骨是一种良好的骨组织工程支架材料。
主要创新点
1.对PDBM与成骨诱导的hADSCs的生物相容性进行了研究。
2.对hADSCs在体外以PDBM为支架的三维培养条件下成骨细胞表型ALP、CON进行了定量检测。
3.成功的进行了hADSCs与PDBM支架裸鼠体内异位构建组织工程化骨。
Background
Bone defects caused by trauma,infection,tumor and so on,are one of the common diseases,and the clinic repair of wide-bound bone defects is still hard to tackle at present.Autogenous bones,allogenic/allogenous bones,and artificial bone substitutes are now usually used as obturation materials for bone defects,which have their respective strong and weak points.Surgeries to get autogenous bones cause a lot of complications,such as pains,blood loss,infection,structure and function destroy of donor sites.In addition,the sources of autogenous bones are relatively less,leading to the limited quantity available.Allogenic/allogenous bones are ready to cause reject reaction and may lead to infection of pathogens,due to their immunogenicity.Though artificial bone substitutes,such as hydroxylapatite and calcium phosphate,can make up the shortcomings of biological source-derived repairing materials,they are weak in bone induction,so their applications are limited merely to bone obturation,bone supporting and bone conduction.Biomaterials combined with growth factors,such as BMP,cannot form complete bone tissue in vivo to satisfyingly repair bone defects, due to the indeterminateness of activity and concentration of the regional BMP. Therefore,preferable repair of bone defects has been the focus of reconstructive surgery todate.
Recent development of research on materials,cytobiology,and molecular biology,has made the features and predominances of tissue or organ reconstruction using tissue engineering methods identified with generally.Furthermore,it is presently received that bone tissue engineering is one of the fields which are probable to be put into practice clinically.
Construction of tissue-engineered bone refers to three key factors,namely seed cells,scaffolds,and methods of construction,in which the former two are nowadays the focal points of research.Osteoblasts are relatively ideal seed cells for bone tissue engineering,however,the deficiency of source impedes their further application.So, it is the key point for further development of bone tissue engineering to look for new cell source.
In 1994,Kaplan et al put forward that the precursor cells of adipose could differentiate into osteoblasts as they discovered heterotopic ossification in the subcutaneous fatty tissues of patients.In 2001,Zuk et al reported the proper cells derived from adipose tissue not only expressed the same surface markers as mesenchymal stem cells(MSCs),but also had the potential to differentiate into osteogenesis,chondrogenesis,adipogenesis,myogenesis and neurogenesis, confirming the cells with stem cell features in adipose tissue.Thus,the concept of ADSCs was introduced.Afterwards,subcutaneous ossification by ADSCs in athymic mice which had immunodeficiency was documented.Research on adipose-derived stem cells(ADSCs) as seed cells has become the hot point of tissue engineering recently.Adipose tissue distributes widespreadly in human bodies,and they can be sufficiently provided from liposuction of plastic surgeries.It was reported that 2×10~8～6×10~8 monocytes could be harvested from 300 ml lipoaspirates.ASDCs are similar as MSCs,which are derived from embryo mesenchymal tissues.It was identified by experiments that ADSCs could undergo long-term culture in vitro,have stable doubling time as well as low level of cell senescence,and they are not significantly different from MSCs speaking of harvest rate of primary culture,cell growth kinetics,cell aging,ability of multi-directional differentiation and gene transfection efficiency.Morever,ADSCs and MSCs are similar in phenotype expression,and can both specifically express protein stro-1.
Research on scaffold materials is one of the important fields of bone tissue engineering.Ideal scaffolds for bone tissue engineering have the characteristics such as good biocompatibility,favorable biodegradation,proper mechanical strength, proper aperture and interval porosity,bioactivity,ability to mediate signal transduction and interaction among seeded cells,and potential of osteoinduction.Now there are two kinds of biomaterials usually used for bone tissue engineering,one is natural bio-derivants,the other is artificial synthetic materials.Compared with other biomaterials,demineralized bone matrix(DBM) has distinctive features and so it attracts the attention of the researchers.Bone tissue-derived DBM can be acquired after the processes of defatting and decalcification,which provide it with satisfactory biocompatibility and bio-degradation,due to its essential component of collagen typeⅠand the the remaining stereo-structure with porosity.However,most of the present DBM materials are completely decalcified homologous bone matrix,they are weak in mechanical strength and degrade relatively fast.In addition,the donors' genders,ages, donor sites and the different process methods could probably affect the quality of DBM.Therefore,the traditional DBM materials cannot meet the demands of bone tissue engineering.Partially demineralized bone matrix(PDBM) materials are acquired by partial decalcification of bone tissue.They are used as scaffolds for bone tissue engimeering as they have comparably lower decalcification degree,enhanced mechanical strength as well as stereo-structure with porosity.Researches on compatibility and interaction between PDBM and ADSCs haven't been documented.
Objective:
To investigate the feasibility of tissue-engineered bone construction by PDBM combined with human ADSCs(hADSCs).
Methods
1.In vitro osteoinduction of hADSCs
(1)Human ADSCs were isolated from human lipoasperates,and were further cultured in vitro.Growth curves of passage 0,1,2,5 were tested.The surface markers of Human ADSCs were detected.
(2)Passage 2 hADSCs cultured with osteogenic medium were set as experimental group,uninduced hADSCs were set as control group.Alkaline phosphatase(ALP) of both groups were stained by BM-Purple method,calcium nodes were stained by Von-Kossa method,immunofluorescence observation was performed to detect collagen typeⅠand osteocalcin(OCN) expression,RT-PCR analysis was performed to test collagen typeⅠ,osteopontin(OPN) and ALP mRNA expression.
2.Compatibility assay of PDBM cultured with hADSCs
(1)Passage 2 hADSCs were osteoinduced and seeded onto PDBM in 4 days, inverted phase cntrast microscope was used to observe hADSCs growth after seeding, surface and transaction of the cell/scaffold construction were observed by SEM in 7 days.
(2)The growth of DIO-labeled hADSCs was observed by Laser Scanning Confocal Microscope(LSCM),cell adhesion rate was tested,ALP activity and OCN secretion were also quantitatively measured using specific assay kits.
3,In vivo construction of tissue-engineered bone by PDBM combined with hADSCs
(1)Isolation and in vitro culture of hADSCs.
(2)PDBM materials were cut into cubes with the dimension of 10mm×5mm×3mm.After radiation sterilization by Co 60,passage 2 hADSCs of 3×10~7/ml were seeded onto the cubes.The cell/scaffold constructions were incubated at 37℃with 5%CO2 for 4h.Then osteogenic medium was added and the constructions were subcultured for 7d.
(3)Sixteen 5-week-old athymic mice were averagedly distributed into two groups, After inhalation anesthesia by diethyl ether,2cm-long transverse incisions were performed on the back of the athymic mice,and subcutaneous gaps were disjuncted towards abdomen bilaterally.Then cell/scaffold constructions were implanted subcutaneously at right side as experimental group,PDBM materials without cells were implanted subcutaneously at left side as control group.The observation duration of the first one was 8w,the second one was 12w.
(4)After sacrifice,paraffin sections H&E staining was performed.
Results
1.Experiment of osteogenic differentiation of human adipose-derived stem cells in vitro
Subcultured hADSCs expanded faster than passage 0.Passage 1,2 and 5 hADSCs have features in common:delitescence was about 24h,logistic proliferative phase lasted for 2～3 days,4～5 days after passage the platform phase began;14 days after passage,as for ALP detection,osteoinduced cells stained positive,the controls stained negative;as for Von Kossa staining to observe ossification,osteoinduced cells stained deep brown,demonstrating positive,and the staining became denser with the increase of culture time,however,the controls stained negative;as for immunofluorescence observation,both of the two groups expressed collagen typeⅠpositively,osteoinduced hADSCs expressed OCN and OPN positively,the controls negatively or weak positively;as for RT-PCR analysis,after 14 days of culture, osteoinduced cells expressed ALP and OPN positively,the controls negatively,both of the two groups expressed collagen typeⅠpositively as positive control group.
2.Empirical Study of Biocompatibility of partially Demineralized Bones Matrix Cultured with hADSCs
Human ADSCs adhesived to PDBM well as detected by SEM,DIO-labeled cells went up in number as culture time increased and could almostly stuff all of the pore gaps of PDBM in 7 days.Quantitative assays for ALP and OCN showed that ALP began to express in the induced cells at the 3rd day,7days later the peak value appeared,and ALP content gradually declined since then;however,ALP content in the non-induced cells maintained at a relatively low level;as culture time increased OCN content gradually rised in both induced and non-induced cells.But OCN content in induced group was significant higher than that of non-induced group.Moreover,as culture time increased,the difference was more clear.
3.In vivo construction of tissue-engineered bone by PDBM combined with hADSCs
As H&E staining shown for the forst group,8w after implantation,newly formed bone trabeculas adhesived to the surface of PDBM like islands.Twelve weeks later for the second group,newly formed bone trabeculas also adhesived to the surface of PDBM like islands,along the fringe of which sequentially aligned a layer of osteoblast-like cells.
Conclusion
1.Human ADSCs can be isolated from human lipoasperates,and were further cultured in vitro.Human ADSCs grow fine in vitro,and passage 2 hADSCs can differentiate into osteoblasts cultured with osteogenic medium.
2.Osteoinduced hADSCs adhere to the surface of PDBM well and grow fine on the surface of PDBM,demonstrating that PDBM has good biocompatibility.Human ADSCs can maintain osteoblast phenotype when subjected to osteoinduction under osteo-culture in vitro.
3.Tissue-engineered bone tissue can be formed in vivo by PDBM combined with osteoinduced hADSCs.It is demonstrated that PDBM is a kind of favorable scaffold biomaterials for bone tissue engineering.
Innovation points
1.The biocompatibility of Osteoinduced hADSCs and PDBM were studied.
2.Quantitative assays for ALP and OCN of osteoblast phenotype of Osteoinduced hADSCs were made by three-dimensional scaffold(PDBM) in vitro.
3.It is the first time to constructe tissue-engineered bone by PDBM combined with hADSCs In vivo.
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