Experimental Study Of Tissue-engineered Bone Constructed By Partially Demineralized Bone Matrix Combined With Human Adipose-derived Stem Cells

BackgroundBone 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).Methods1.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 vitroSubcultured 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 hADSCsHuman 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 hADSCsAs 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.Conclusion1.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 points1.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.