Comparison of menchymal stem cells from bone marrow and adipo tissue for bone regeneration in a critical size defect of the sheep tibia and the influence of platelet-rich plasma
Philipp Niemeyer a ,Katharina Fechner b ,Stefan Milz c ,Wiltrud Richter b ,Norbert P.Suedkamp a ,Alexander T.Mehlhorn a ,Simon Pearce c ,Philip Kasten b ,d ,*
a
Department of Orthopedic Surgery and Traumatology,Freiburg University Hospital,Freiburg,Germany b
Department of Orthopedic Surgery,Heidelberg University Hospital,Heidelberg,Germany c
AO Rearch Institute,AO Foundation,Davos,Switzerland d
Department of Orthopedic Surgery,University of Dresden,Dresden,Germany
a r t i c l e i n f o
Article history:
Received 17December 2009Accepted 13January 2010
Available online 11February 2010Keywords:
Bone,tissue engineering Bone regeneration Platelet
Growth factors Stem cell
Adipo tissue
a b s t r a c t
Aim of the prent study was to compare the osteogenic potential of bone marrow derived menchymal stem cells (BMSC)and adipo-tissue derived stem cells (ASC)and to evaluate the influence of platelet-rich plasma (PRP)on the osteogenic capacity of ASC in a large animal model.
Ovine BMSC (BMSC-group)and ASC (ASC-group)were eded on mineralized collagen sponges and implanted into a critical size defect of the sheep tibia (n ¼5each).In an additional group,platelet-rich plasma (PRP)was ud in combination with ASC (PRP-group).Unloaded mineralized collagen (EMPTY-group)rved as control (n ¼5each).Radiographic evaluation was performed every 2weeks,after 26weeks histological analysis was performed.
Radiographic evaluation revealed a significantly higher amount of newly formed bone in the BMSC-group compared to the ASC-group at week 10and compared to EMPTY-group from week 12(all p <0.05).A superiority on radiographic level concerning bone formation of the PRP-group versus the empty control group was found (p <0.05),but not for the ASC-group.Histological analysis confirmed radiographic evaluation finding analogous significances.
In conclusion,ASC em to be inferior to BMSC in terms of their osteogenic potential but that can partially be compensated by the addition of PRP.
Ó2010Elvier Ltd.All rights rerved.
1.Introduction
Menchymal stem cells (MSC)are an attractive cell population for regeneration of menchymal tissue such as bone.Various studies have been published demonstrating the bone-building capacity of menchymal stem cells and even their ufulness in treating critical size bone defects.Most of the studies were conducted with MSC derived from bone marrow (BMSC).However,other sources have recently been described to contain corre-sponding cell populations.MSC with characteristics similar to bone marrow derived cells can be obtained from human adipo tissue
[8],ligaments [9],and lung [10].MSC derived from adipo tissue (ASC),in particular,are considered to be an attractive alternative to MSC from bone marrow,becau of their abundant availability and excellent expansion and proliferation capacities.Although ASC have now been sufficiently characterized,and their differentiation potential has been described extensively,there is still a lack of in vivo studies comparing the regenerative potential of ASC with tho of BMSC in an orthotopic large animal model.The hypothesis was that ASC have an equivalent osteogenic potential as BMSC.
In addition,the influence of platelet-rich plasma (PRP)on bone healing using adipo-tissue derived menchymal stem cells (ASC)was subject of the prent study.PRP has been of increasing interest in recent years and the data available in literature are controversial.While some studies report a significant improvement of bone healing in prence of PRP [1–6],other studies were not able to detect any positive influence [7–10].Nevertheless,the influence of PRP on menchymal stem cells has only been investigated on cells derived from bone marrow (BMSC).Since adipo-derived stem
*Corresponding author at:Division of Shoulder and Elbow Surgery and Sports Medicine,Department of Orthopaedic Surgery,Dresden University Hospital Carl Gustav Carus,University of Dresden,Fetscherstras 74,01307Dresden,Germany.Tel.:þ493514585006;fax:þ493514584376.
E-mail address:philip.kasten@uniklinikum-dresden.de (P.
Kasten).Contents lists available at ScienceDirect
Biomaterials
journal homepage:/loca
te/biomaterials
0142-9612/$–e front matter Ó2010Elvier Ltd.All rights rerved.doi:10.1016/j.biomaterials.2010.01.085
Biomaterials 31(2010)3572–3579
cells obviously behave differently in a biological environment,the influence of PRP on ASC has to be evaluated parately.Accordingly, the cond hypothesis was that the addition of PRP to ASC is beneficial for bone healing.
2.Materials&methods
2.1.Isolation and expansion of ovine MSC from bone marrow aspirates
Ovine MSC were obtained from the iliac crest under local anesthesia and procedural dation for a later autogenous transplantation.The anterior superior iliac spine was aspirated yielding between15and40ml bone marrow.BMSC were isolated as published elwhere[11]with minor variations at a density of1Â105/cm2. Briefly,bone marrow mononuclear cells were obtained by Biocoll density gradient centrifugation(d¼1.077g/cm3;Biochrom)and plated infibronectin-coated tissu
e cultureflasks(Nunc).The expansion medium ud was58%low-gluco DMEM (Cambrex),40%MCDB201(Sigma),2%FCS(Stemcell Technologies,Inc.),supple-mented with2m M L-glutamine,100U/ml Pen/Strep,ITS,linoleic acid,10n M dexa-methasone,0.1m M L-ascorbic-acid-2-phosphate(all from Sigma),PDGF-bb and EGF (10ng/ml each,R&D Systems).
2.2.Isolation of ovine MSC from adipo tissue
Adipo tissue(starting quantity5–7g)for the autogenous isolation of ovine MSC was obtained from experimental animals under local anesthesia(2–5ml lidocaine s.c)and analgodation(2mg/kg ketamine,0.05mg/kg,, circa5–10m g fentanyl/kg i.v.).A lumbar paravertebral excision site was chon.After a3–5cm skin incision,the subcutaneous fat tissue was discted and removed using scalpel and scissor,and placed directly into a correspondingly labeled falconflask containing phosphate buffered saline(PBS)with20mg/ml bovine rum albumin (BSA)and1%penicillin/streptomycin.Ovine MSC were obtained by enzymatic digestion with collagena according to standing protocols[12,13].The adipo tissue was then washed twice with PBS containing20mg/ml BSA.The samples were then placed in petri dishes to reduce the adipo tissue to portions approximately 0.5cm in size and to disct and removefibrous material and blood vesls.A 0.6mg/ml collagena solution(collagena type B(Roche diagnostics,derived from Clostri
dium hydrolyticum)with a specific activity of>0.15U/mg)was obtained by dilution with Krebs–Ringer buffer containing HEPES and the adipo tissue was incubated with an equivalent volume of collagena solution and agitated lightly at 37 C for90min.Undigested tissue and endothelial cell aggregates were largely removed by subquentfiltration through a polypropylene membrane with a pore size of150m m.After centrifugation of the cells at600Âg for10min at room temperature,the supernatant together with the lipid layer were discarded and the cell pellet was washed twice with PBS.The cells were resuspended in expansion medium and eded into25cm2cell culture bottles(Nunc,Denmark).Cells were expanded at37 C and6%CO2with medium according to Verfaille as published previously[11,14].One or two days after eding the adherent cells were thoroughly rind with PBS to remove non-adherent cells from the culture.
形容狗的词语
2.3.Verification of the MSC character of BMSC and ASC
To verify their MSC character,BMSC as well as ASC were successfully differen-tiated into bone,cartilage and fat according to standard protocols[11,15].For this purpo,after differentiation into different lineages,cells were stained for collagen type II(after chondrogenic differentiation;antibody:ICN,Aurora,Ohio,USA)as well as with van Kossa(after osteogenic differentiation;Sigma,Taufkirchen,Germany) and with oil red staining(after adipogenic differentiation;
Sigma,Taufkirchen, Germany).All stains were performed according to the manufacturer’s protocols.For both cell types,cells from passages2–4(corresponds to16–20doublings)were ud for experiments.
2.4.Seeding and3D cultivation on mineralized collagen
Two rectangular pieces of mineralized collagen(total volume5cm3corre-sponding to the bone defect size)per animal were placed into individual wells of a Petri dish,with care being taken to e that they were not in direct contact with the surrounding well border.The mineralized collagen sponge(Healos aˆ,DePuy,USA) ud in this study is a soft lyophilized,3-dimensional matrix constructed of cross-linked collagen-1fibers coated with non-crystalline hydroxyapatite.The principal components of the matrix are type I bovine collagen and non-ceramic hydroxyap-atite[Ca10(PO4)6(OH)2].Pore sizes vary from4m m to200m m.The overall porosity is >95%.The Ca/PO4ratio is1.67.The mineralized collagen sponge contains approxi-mately30%mineral by weight.All matrices were loaded with2Â107cells as pub-lished earlier[16].In brief,trypsinized MSC were resuspended and2Â107cells per animal were applied to each of the matrices(viability>95%as determined by trypan blue staining).After one hour of dimentation,the dishes werefilled up with expansion medium[11]and incubated at37 C until transplantation.In all cas the transplantation was performed within24h after
eding.2.5.Preparation and characterization of platelet-rich plasma
Allogeneic human leukocyte-depleted PRP was obtained from a local blood bank.The PRP was prepared at room temperature by centrifugation(20min, 150Âg)of a pool of3buffy coats(mean age29,standard deviation(SD)7)derived from whole blood donations that were1:10anticoagulated with acid-citrate-phosphate-dextro(Compoflex,Frenius HemoCare,Bad Homburg,Germany). After a cond centrifugation at2200Âg for15min at room temperature,a mean platelet count in the leukocyte-depleted PRP to1.0Â109/ml corresponding to a4–5 fold increa of the platelet count in whole blood was adjusted.The platelet-rich supernatant was leukocyte-depleted viafiltration(Frenius Bio P Plus,Frenius HemoCare).The residual leukocytes were less than1Â106per unit of PRP.PRP from a total of3donors was mixed to achieve reprentative and standardized values of the growth factors.The PRP was frozen2–3h after donation in50ml falcons at À20 C and stored until needed.To characterize the content of the growth factors in the PRP,the PDGF-AB and TGF-b1contents of ten donors were quantified by a commercial enzyme-linked immunoassay(ELISA)(Quantikine Human R&D Systems,Minneapolis,USA).The platelets were degranulated to relea the growth factors by freezing and thawing[6].The content of PDGF-AB was185.67ng/ml and of TGF-b197.11ng/ml.
The construct of the PRP-group received40ml of PRP just before implantation into the defect and was activated with5ml10%CaCl solution with20IE thrombin (Tissucol Duo(Baxter)/ml CaCl).
2.6.Animal model and group size
举国上下Five animals were assigned to each group and euthanized6months post-operatively.There were four different groups in this experiment(e Table1). Whereas unloaded mineralized collagen rved as control in the EMPTY-group,in the BMSC-group2Â107autogenous ovine BMSC were transplanted on mineralized collagen,and in the ASC-group collagen scaffolds were eded with the same amount of autogenous ovine ASC.In the PRP-group,ASC were applied in the same fashion as in the ASC-group with the exception that additionally40ml activated PRP was applied into the defect after positioning of the cell-scaffold construct.Three year old,approximately65kg heavy female non-pregnant Swiss alpine sheep were ud for this study.Prior to the beginning of the study,all animals were routinely de-wormed with0.2mg/idectin(CydectinÒ,Wyeth Pharmaceuticals, Mu¨nster,Germany)and0.2mg/doramectin(DectomaxÒ,Pfizer,Karlsruhe, Germany).The animals were fasted24–36h preoperatively with ad libitum access to drinking water.Premedication consisted of0.3mg/kg i.v.diazepam(ValiumÒ, Roche)and0.08mg/kg i.v.butorphanol(MorphasolÒ,Gra¨ub,Bern,Switzerland).
2.7.Animal surgery
For the operation the animals were put under general anesthesia and prepared for surgery as published previously[17].In the mid diaphysis(7cm measured from the medial malleolus of the tibia)the bone was marked over a length of three cm. After that,a CF-PEEK plate(38%carbonfibre reinforced polyether-ether-ketone plate,according to its characteristic shape later called‘Snakeplate’,custom made by icotec AG,Altsta¨tten,Switzerland)was placed and the holes1,2,6and7were drilled.Holes3,4and5were left empty as they corresponded to the defect zone.The plate was removed and a2.7mm custom-made spacer was placed andfixated with a monocortical screw in the middle of the pre-determined defect zone.A full diameter ction of the bone was cut out using an oscillating saw.Care was taken that all of the periosteum was removed.In addition,the periosteum was removed at both sides of the osteotomy for additional3mm.After placing the Snakeplate again the threads were cut,measured,and screws,made of the same material,but also including tantalumfibers,which makes them visible on radiographs,were intro-duced.The screws were tightened with a force of1.5N m.Following that,a7hole LCP plate(Synthes GmbH,Oberdorf,Switzerland)wasfixed to the cranial aspect of the tibia(Synthes GmbH,Oberdorf,Switzerland).Care was taken that the
plates
Table1
Different experimental groups ud in the current study,survival time for all animals
P.Niemeyer et al./Biomaterials31(2010)3572–35793573
were in a 90 angle to each other,that all screws were placed bicortically,and that the screws did not interfere with each other.The defect was a full-size 3cm mid diaphyal defect with the periosteum completely removed.2.8.Postoperative management and monitoring
Postoperative analgesia was provided by intramuscular injection of carprofen (Rimadyl Ò4mg/kg)an
d subcutaneous injection of buprenorphine (Temgesic Ò0.01mg/kg)at the end of surgery.The sheep also received buprenorphine three times daily for 3days and carprofen once a day for five days.No antibiotics were admin-istered.During the duration of the study,the sheep were checked every 24h by an experienced animal caretaker.For eight postoperative weeks,the sheep were kept in suspension slings which allowed them to stand and to bear full weight on all legs,but protects them from high loading forces,especially during lying down.After that,the sheep were kept in single boxes.After six months the sheep were euthanized by means of an intravenous overdo of barbiturate (pentobarbital,Vetanarcol Ò).After-wards,both hind legs were exarticulated at the knee and the tibia was discted,the implants were removed,and the samples were procesd and analyzed.2.9.Radiographic analysis
Correct positioning of the osteosynthesis was confirmed by radiography immediately postoperatively,then every two weeks,and also post mortem.A digital radiographic suite was ud (Philips,Trauma Diagnost).The animals were dated using 0.03mg/kg (Domodan Ò,Pfizer,Karlsruhe)and fixated by an assistant.Biplanar radiographs were performed:anterior/posterior (48kV/15mA s)and laterolateral (53kV/32mA s),for which the sheep were positioned in a lateral recumbent position.Quantification of osteoneogenesis was performed usi
ng a method developed by our group which was compared to alternative methods with regard to reliability,variability and objectivity in a previous study [18].The latero-lateral radiographs were analyzed with public domain computer software (GIMP,GNU General Public Licen).Using this software,it is possible to quantify the osteoneogenesis bad on histogram analysis.Quantification of newly formed bone is performed as follows:A rectangular area of pre-defined size (300Â300pixels)is lected to define the ‘‘region of interest’’(ROI).Then,the pixel zone reprenting ossification is defined:the lower end of the range is created using 3individual measurements of the connective tissue using the color lector pipette tool.This creates the lower limit of the interval defined as the ossification zone.Corre-spondingly,pixel values of cortical bone are ud to t the upper limit.The entire ossification zone was divided into three equally sized regions defined as beginning,good,and very good ossification,thus providing improved and simplified asss-ment.The histogram function was then ud to asss the pixel values of the three regions within the bone defect.The regions of ‘‘good’’and ‘‘very good’’ossification were considered newly formed bone for the prent study.2.10.Histological evaluation
The explanted tibia samples were split longitudinally,and one half was embedded in methylmethacrylate (MMA,Fluka,Switzerland)for histological eval-uation as published previously [17].
In brief,the samples were fixed in 70%meth-anol,dehydrated using a ries of increasing concentrations of alcohol (70%,96%und 100%ethanol),and embedded in methylmethacrylate monomer (MMA).After polymerization the samples were cut longitudinally using a sawing microtome (Leica SP 1600).Two to three centimeters of the original bone tissue proximal and distal to the defect were included in the sample.Giemsa–Eosin staining was per-formed to enable the morphological analysis of the non-decalcified polished MMA ctions with Giemsa solution (15%)and eosin counterstaining as published previ-ously [17].
2.11.Fluorescence marking
Fluorescent in vivo marking was performed during the postoperative pha of the study at lected time points to allow analysis of new bone formation.Animals were marked with 60mL of calcein green injected subcutaneously in the eighth week and with 60mL of xylenol orange in the tenth week.The dyes are safe for the animals and are incorporated into the bone formed at the time of bioavailability.This enables time-locking when the bone samples are assd histologically using a fluorescent light source (Leica M165FC,450nm).2.12.Histological evaluation
Special attention was paid to heterotopic ossifications and signs of inflammation or potential tissue r
ejection reaction during the descriptive evaluation.The locali-zation of newly formed bone was also noted.Quantitative evaluation was performed in accordance to the Mosheiff Score [19]and the Werntz Score [20],both of them established for asssing experimental bone defects.Furthermore,the area of newly formed bone was analyzed with public domain image processing software GIMP (GNU General Public Licen)(in equivalent fashion as the radiologic evaluation mentioned above)and put into relation to the total area of the bone defect.To calculate the Mosheiff Score [19]ranges of interest (ROI)were generated that could be superimpod on the images and which demarcated nine small fields of pre-defined size (25mm 2,three proximal,three medial,and three distal).The ROIs was then superimpod on the scanned digital histological images and each of the nine small areas was independently given a score ranging from zero (no ossification)to two (good ossification).The values were assd individually for each slice and then added to give a reprentative measure of total ossification.In the Werntz score [20],different qualities of the bone regions are taken into consideration and points are given for each quality.Zero to five points can be given for the category ‘‘bone formation’’and zero to four points each for the categories ‘‘union’’and ‘‘remodel-ling.’’The individual categories are analyzed independently and also after addition of the points.
2.1
3.Statistical evaluation如何设置锁屏时间
After data collection,parate data existed from radiographic follow up and histological asssments.Radiographic data from 14time points existed at evalua-tion.There was data from histological evaluation corresponding to the three different methods of quantification.All data and measurements from the three test groups pasd tests for normal distribution,thereby fulfilling prerequisites for subquent statistical analys.The results were examined by a multifactorial variance analysis (ANOVA).Afterwards,differences between the independent vari-ables were checked in Turkey post-hoc tests.Alpha error was conquently adjusted,p-values <0.05were taken to be significant.All tests were two tailed.In addition,a paired t-test was also performed to examine radiological differences for each group over the cour of the experiment.Statistical analysis was performed using the SPSS statistics package version 15.0.
3.Results
3.1.Radiographic quantification of newly formed bone
A significant superiority of the BMSC-group versus the ASC-group could be detected from week 10until the end of the study period (week 26)(Figs.1and 2).In addition,significantly more bone formati
on could be detected in the BMSC-group compared
to
Fig.1.Reprentative radiographs 26weeks after surgery (lateral view)of the best animal of each group (from left to right):ASC-group,EMPTY-group,PRP-group;BMSC-group.
P.Niemeyer et al./Biomaterials 31(2010)3572–3579
安平桥3574
the EMPTY-group between weeks 12and 26.Concerning the influence of PRP,a trend to more bone f
炸馒头片
ormation in the PRP-group compared to the EMPTY-group was detected between week 20and week 26(p-values between 0.1and 0.05),but this obrvation lacked statistical significance (Fig.3).
Compared to postoperative percentage of bone in the defect,in the BMSC-group,significantly more bone compared to immediately postoperative obrvations (day 1)was detected from week 8until the end of the study period (all p-values <0.05,Fig.4).While in the ASC-group,no significant bone formation compared to day 1could be detected at any of the time points investigated,in the PRP-group,significantly larger amounts of bone were found in weeks 22and 26(p-values <0.05).
deepin系统
3.2.Histological evaluation
Analysis of all histological samples of the BMSC-group and of 2out of 5samples of the PRP-group exhibited bridging of the defect.In none of the animals that were assigned either to the
EMPTY-group or to the ASC-group a bridging of the defect was achieved.Reprentative histological samples of different treat-ment groups are given in Fig.5.Detailed descriptive evaluation of the histological samples revealed the prence of osteoid and ‘‘bone lining cells’’as histological characteristics of mainly direct ossification (Fig.6).Partially,also histological signs of indirect ossification (enchondral ossification)could be detected.In the majority of the animals,bone formation
started at the proximal and distal osteotomy.Bone in the defect emed to be less mature and this was mainly obrved in the animals in which bridging occurred late within the study period.In addition,fluorescence marking revealing an earlier fusion in the PRP-group compared to the BMSC-group.This was clearly visible in tho animals in which osus bridging occurred (Fig.7).In all animals treated with cell-loaded implants (all groups except for the EMPTY-group)areas of newly formed bone without contact to the edges of the osteotomy zone were found.In the ASC-group areas of cells with large vacuoles similar to a adipogen phenotype were obrved (Fig.8
).
框框里面打勾的符号Fig.2.Quantitative radiographic asssment of bone formation in the defect zone in the clinical cour following surgery comparing BMSC,ASC and the empty-group:The BMSC-group (black columns)shows significant more bone formation from week 10to the end of the study period compared to the ASC-group (white columns)and from week 12to week 26compared to the EMPTY-group:(grey columns);significant differences (p <0.05are indicated by
‘‘*’’).
Fig.3.Quantitative radiographic asssment of bone formation in the defect zone in the clinical cour following surgery comparing PRP,ASC and the empty-group:In the mean,the PRP group (black columns)shows more bone formation compared to the ASC-group (white columns),and compared to empty scaffolds (EMPTY-group:grey columns);nevertheless,probably due to a heterogeneity between different individuals from the PRP-group (reprented by a high standard deviation),this obrvation lacked of statistical significance.
P.Niemeyer et al./Biomaterials 31(2010)3572–35793575
3.3.Quantitative analysis of histology
For quantitative histological asssment of newly formed bone,imaging software describing the percentage of newly formed bone in the defect was ud as well as two established mi-quantitative scoring systems.All three methods demonstrated analogous results.Concerning the quantitative determination of newly formed bone,at the end of the study a period with significantly higher amount of bone was detected in the BMSC-group compared to the ASC-group (p ¼0.010).Concerning the influence of PRP,a larger amount of bone in the PRP-group compared to the ASC-group was detected as a trend but failed to reach statistical significance (p ¼0.084).In additi
on,animals treated with autoge-nous BMSC showed higher amounts of bone compared to unloaded controls (p ¼0.023).Results are given in Fig.9part A.Using the Werntz score,superiority of BMSC compared to ASC was found as a trend (p ¼0.080).The sub-score ‘‘union’’revealed a superiority of BMSC versus ASC (p ¼0.049,Fig.9part B).According to the mi-quantitative scoring system ud by Sarkar,significantly more bone formation was detected in the BMSC-group versus the EMPTY-group (p ¼0.032)and the ASC-group (p ¼0.042,Fig.9part C).4.Discussion
In vivo data regarding the osteogenic potential of adipo menchymal stem cells (ASC)to heal bone defects remain scarce [21–24].The cells are enormously attractive for clinical u becau of their ready availability and easy accessibility.Prior to clinical u,however,it must be proven that their osteogenic potential is not inferior to that of bone marrow cells.At the moment,there are few experimental studies using bone defect models addressing this issue [22,25,26].The studies suggest that undifferentiated ASC lead to functionally inferior regenerated tissues compared to MSC derived from bone marrow or periost.With the exception of the Peterson study,which examined the efficacy of BMP-2transfected ASC,the results of the studies are of only limited relevance for various reasons.One problem was that in the study of Hui et al.the cells were applied within a fibrin glue suspension [25].From our perspective,this approach is limited concerning its lack of biomechani
cal stability which is needed in a weight-bearing bone defect.Another problem is that a rabbit model which has a high rate of spontaneous healing was employed,and furthermore the defects were no critical size bone defects,but rather partial defects only [25].In the study by Hayashi et al.a small
animal model (rat defect model)was ud,which only allows a very limited extrapolation of results to the human patients situation and therefore must be regarded inferior to a defect model in a large animal [26].Nonetheless,the studies alert the reader to possible problems which could ari with the u of adipo stem cells and thereby justify the parate and comparative analysis of the regeneration potential of the cells with autogenous BMSC.The in vivo bone defect model studies also showed that the initially comparative studies,which demonstrated esntially identical properties of adipo and bone marrow stem cells at the in vitro level [27,28]and after heterotopic in vivo transplantation [29],cannot uncritically be transferred to the regeneration capacity of orthotopic
bone.
Fig.4.Quantitative evaluation of newly formed bone in the defect (in %),with regard to differences compared to ‘‘day 0’’in dependence of group assignment.Percentage of defect filled with bone in %is visualized from day 0(left column)to week 26(right column)of every group.Significances (p <0.05)compared to day 0are visualized by black
columns.
Fig.5.Reprentative histological slides of animals assigned to different treatment groups (overview).
P.Niemeyer et al./Biomaterials 31(2010)3572–3579
醒目的近义词
3576
