Novel therapeutic core–shell hydrogel scaffolds with sequential delivery of cobalt and bone morphogenetic protein-2for synergistic bone
regeneration
Roman A.Perez a ,b ,Joong-Hyun Kim a ,b ,Jennifer O.Buitrago a ,b ,Ivan B.Wall b ,c ,Hae-Won Kim a ,b ,d ,?
a
Institute of Tissue Regeneration Engineering (ITREN),Dankook University,Cheonan 330-714,South Korea
b
Department of Nanobiomedical Science &BK21PLUS NBM Global Research Center for Regenerative Medicine,Dankook University,Cheonan 330-714,South Korea c
Department of Biochemical Engineering,University College London,Torrington Place,London WC1E 7JE,UK d
Department of Biomaterials Science,College of Dentistry,Dankook University,Cheonan 330-714,South Korea
a r t i c l e i n f o Article history:
Received 16March 2015
Received in revised form 5May 2015Accepted 1June 2015
Available online 6June 2015Keywords:Osteogenesis Angiogenesis
Therapeutic scaffolds Bone formation Sequential delivery
a b s t r a c t
Enabling early angiogenesis is a crucial issue in the success of bone tissue engineering.Designing scaffolds with therapeutic potential to stimulate angiogenesis as well as osteogenesis is thus considered a promising strategy.Here,we propose a novel scaffold designed to deliver angiogenic and osteogenic factors in a sequential manner to synergize the bone regeneration event.
Hydrogel ?brous scaffolds comprised of a collagen-based core and an alginate-based shell were constructed.Bone morphogenetic protein 2(BMP2)was loaded in the core,while the shell incorporated Co ions,enabled by the alginate crosslinking in CoCl 2/CaCl 2solution.The incorporation of Co ions was tunable by altering the concentra-tion of Co ions in the crosslinking solution.The incorporated Co ions,that are known to play a role in angiogenesis,were released rapidly within a week,while the BMP2,acting as an osteogenic factor,was released in a highly sustainable manner over several weeks to months.The release of Co ions signi?cantly up-regulated the in vitro angiogenic properties of cells,including the expression of angiogenic genes (CD31,VEGF,and HIF-1a ),secretion of VEGF,and the formation of tubule-like networks.However,BMP2did not activate the angiogenic processes.Osteogenesis was also signi?cantly enhanced by the release of Co ions as well as BMP2,characterized by higher expression of osteogenic genes (OPN,ALP,BSP,and OCN),and OCN protein secretion.An in vivo study on the designed scaffolds implanted in rat calvarium defect demonstrated signi?cantly enhanced bone formation,evidenced by new bone volume and bone density,due to the release of BMP2and Co ions.This is the ?rst study using Co ions as an angio-genic element together with the osteogenic factor BMP2within scaffolds,and the results demonstrated the possible synergistic role of Co ions with BMP2in the bone regeneration process,suggesting a novel potential therapeutic scaffold system.Statement of Signi?cance
This is the ?rst report that utilizes Co ion as a pro-angiogenic factor in concert with osteogenic factor BMP-2in the ?ne-tuned core-shell hydrogel ?ber scaffolds,and ultimately achieves osteo/angiogenesis of MSCs and bone regeneration through the sequential delivery of both biofactors.This novel approach facilitates a new class of therapeutic scaffolds,aiming at successful bone regeneration with the help of angiogenesis.
ó2015Acta Materialia Inc.Published by Elsevier Ltd.All rights reserved.
1.Introduction
Recent advances in bone tissue engineering have focused on developing therapeutic scaffolds with the capacity to load bioactive molecules and deliver them in a controllable and sustain-able manner [1].The in vivo conditions under which damaged tissues are repaired basically require multiple types of bioactive molecules and biological ingredients with their sequential involvement at the correct time and with effective doses [2].Some key elements have long been identi?ed to play key roles in bone repair and regeneration.For instance,bone morphogenetic
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?Corresponding author at:Institute of Tissue Regeneration Engineering (ITREN),Dankook University,Cheonan 330-714,South Korea.Tel.:+82415503081.
E-mail address:kimhw@https://www.doczj.com/doc/fa11287507.html, (H.-W.Kim).
proteins(BMPs)are one of the most ef?cient bioactive molecules that stimulate osteogenic induction,in orthotopic sites and even in ectopic locations[3].Despite the importance of osteogenic induction,successful bone regeneration requires early stage angio-genesis,and this is particularly important when the defect dimen-sion is large and/or tissue engineered cells are engaged.The secretion of angiogenic factors,recruitment of blood vessel-forming endothelial cells and ultimately the stimulation of vascu-lar network formation are key events taking place at the early stage of bone repair and regeneration[4].
Therefore,strategies to improve those initial angiogenic events, accompanied by the stimulation of osteogenesis at a much later stage,have recently been signi?cantly pursued.Noteworthy exem-plar studies include the use of angiogenic factors such as vascular endothelial growth factor(VEGF)and basic?broblast growth factor (bFGF)at the early phase,while osteogenic molecules,including BMP2,BMP7,and dexamethasone are designed to be delivered at a much later stage[5,6].The success of this strategic concept of‘se-quential drug delivery’is largely dependent on the design of scaf-folds.The incorporation of microparticles within scaffolding matrices and the layered structuring of scaffolds are some key examples.In this sense,PLGA microspheres loaded with BMP2 were embedded in polypropylene scaffolds that rapidly released VEGF($3days)and then facilitated more sustained release of BMP2over50days[6].The same effect was achieved using a dif-ferent approach,whereby sequential delivery of VEGF and BMP2 was possible through the layering of polyelectrolyte?lms.In order to achieve this,a degradable tetra layer with a poly(b-amino ester)/polyanion/growth factor/polyanion structure was prepared in sequential layers,enabling sustained release of BMP2over 2weeks,whereas the VEGF within8days[7].
Recently,the authors designed a core–shell structured?brous scaffold made of a collagen core and an alginate shell using a co-concentric nozzle design[8,9].The core–shell?brous scaffolds have been demonstrated to be useful platforms for dual drug deliv-ery[8]as well as cell encapsulation[9].The core–shell structure enabled sequential release of two different biomolecules,which were incorporated either in the core or in the shell part,and the release rate was highly dependent on the molecular diffusion rate. Furthermore,for targeting hard tissues,the incorporation of cal-cium phosphate self-setting powders substantially improved the mechanical properties and slowed down the protein release behav-iors,implying usefulness in long-term delivery of therapeutic molecules.Besides their use in drug delivery,the cells encapsu-lated within the core part were shown to survive,proliferate,and properly adopt osteogenic differentiation under osteogenic envi-ronments.This also translated to excellent performance in vivo, suggesting a possible vehicle for cell delivery.In particular,the sys-tem is based on the use of alginate as the shell,which becomes hardened in contact with divalent cations,such as CaCl2,preserv-ing the core–shell structured scaffolds[8].
On the basis of the core–shell?brous scaffolds,in this study we designed a sequential drug delivery system with angiogenic and osteogenic potential,which is ultimately effective for bone regen-eration.We utilized cobalt(Co)ions as the pro-angiogenic factor. The rationale of using Co is that it has been used to induce hypoxia by blocking the degradation of hypoxia inducing factor(HIF)a1 [10].HIF a1is a transcription factor in the oxygen sensing pathway activated in hypoxic environments and is able to alter gene expres-sion and enhance angiogenesis[11].A recent study utilizing Co has demonstrated its considerable effect on angiogenesis[12]. Therefore,we hypothesized that Co incorporated within the core–shell?brous scaffolds,particularly in the shell part,would be effective in the initial angiogenesis phase.Importantly,the Co ions can be elegantly incorporated within alginate in partial replacement of Ca ions as an alternative cross-linking divalent ionic source[13,14].In this way,we incorporated Co ions within the shell part in the course of alginate hardening in CoCl2/CaCl2 solutions while delivering BMP2in the core part.Fig.1a illustrates schematically the current design of the core–shell?brous scaffolds to sequentially deliver Co/BMP2.
To the best of our knowledge,this co-usage and co-delivery of Co with an osteogenic factor(BMP2)has not yet been reported. While excessive uses of Co may induce cell death,small concentra-tions used in the therapeutic window(reported to be less than 100l M)are expected to have profound positive effects[15,16]. We report this novel design of Co/BMP2sequential delivering scaf-fold,in terms of the process tools,effects on cellular angiogenesis and osteogenesis,and in vivo ef?cacy in bone regeneration.
2.Materials and Methods
2.1.Core–shell?brous scaffold fabrication
For the composition of core and shell materials,we used collagen-and alginate-based hydrogels which also incorporate dif-ferent amounts of self-setting calcium phosphate powder (a-tricalcium phosphate(a-TCP)).The a-TCP powder was obtained by sintering a mixture of calcium hydrogen phosphate(CaHPO4, from Sigma–Aldrich#C7263)and calcium carbonate(CaCO3,from Sigma–Aldrich#239216)at1400°C and subsequent quenching [17,18].The sintered powder was milled in a planetary mill and added with2wt.%hydroxyapatite(HA,from Alco#1.02143)crys-tallites as a seed for the phase-transformation of cement(from a-TCP into HA).The a-TCP powder showed a median particle size of 5.2l m as determined from laser diffraction(Malvern, APA5001SR),and a speci?c surface area of1.23m2/g as measured by N2adsorption–desorption by the BrunaueràEmmettàTeller (BET)method(Quadrasorb SI,Quantachrom Instruments,Ltd., Boynton Beach,FL).Na-alginate(from Sigma–Aldrich#A2158) with an M/G ratio of1.67and a molecular weight of50kDa was dissolved at3%in water.The collagen solution(rat tail type I col-lagen at a concentration of2.05mg/ml,from First Link)was pre-pared by mixing1ml of the collagen solution with100ml of10 Dulbecco’s modi?ed Eagle medium and a proper amount of1N sodium hydroxide which is proper to adjust pH at7.0for subse-quent gelation.
A co-concentric nozzle(inner23G and outer17G)was speci?-cally designed and used to produce core–shell structured?brous scaffolds.Each solution with a speci?c composition of10%a-TCP plus alginate or50%a-TCP plus collagen was separately fed into the outer and inner syringe,respectively.Each syringe was attached to an injection pump connected through a microtube. Core–shell structured collagen-alginate?ber(0.3ml)was then injected at a rate of50ml/h through the co-concentric nozzle within a bath containing150mM of divalent ions(Ca2+or Co2+) for1min.The concentration of ions was optimized for the gelation of alginate shell phase while preserving a good cell viability of the ?ber scaffolds[8,9].Calcium chloride(CaCl2,from Sigma–Aldrich #223506)and cobalt chloride(CoCl2,from Sigma–Aldrich #232696)were used as the source of Ca and Co ions,respectively. Different crosslinking solutions are summarized in Table1.
2.2.Loading and release of Co ion and BMP2
2.2.1.Co ion loading and release
The core–shell scaffolds prepared in the Co ion-containing crosslinking solution incorporate Co ions due to a diffusion of Co through the alginate network.Furthermore,the incorporated Co ions vary depending on the content of Co ions within the crosslink-ing solutions.After preparation of the core–shell scaffolds
296R.A.Perez et al./Acta Biomaterialia23(2015)295–308
crosslinked in different solutions,the release of Co ions from the scaffolds was analyzed.Each scaffold sample was placed in each well of 24-well plates containing 1.5ml of distilled water.After 1,3,and 6days,the medium was replaced.The supernatant was
analyzed to measure the amount of Co ions released from the sam-ple using the Co quanti?cation kit (BioVision,Cobalt Colorimetric Assay kit),following the manufacturer’s guidelines.After the col-orimetric reaction,an absorbance was read at 475mm using a spectrophotometer.Data were analyzed in triplicate (n =3).2.2.2.BMP2loading and release
BMP2protein was obtained as previously described [19].Brie?y,the cDNAs of the BMP2were ampli?ed from an adult human cDNA library.According to the GenBank sequence (GeneBank accession number NM002006),a pair of primers,50-G AAGATCTGCCAAACACAAACAGCGG-3and 50-AACTGCAGATCTGTCT TTTCTACCGCTGGACACCCACAACCCTC-30,were designed and used.The PCR products were cloned into pBAD/His A (Invitrogen,Carlsbad,CA)in-frame using the NH 2-terminal 6X His tag.
The
illustration showing a current study aim to deliver Co and BMP2cofactors in the core–shell ?brous scaffold system.a designed concentric nozzle into a CoCl 2/CaCl 2solution,become a core–shell ?brous scaffold by the hardening of incorporated.The Co incorporated in the outer shell and the BMP2loaded in the inner core are to be released an early phase of blood vessel formation as well as the osteogenesis by BMP2at a later stage of bone formation.(b)from optical microscopy and SEM.Core and shell parts are noted in images.
Table 1
Crosslinking solutions used for the core–shell formation of collagen-alginate thera-peutic scaffolds,where the concentration of divalent cations of Ca and Co was varied to crosslink alginate as well as to incorporate Co ions.CaCl 2(mM)CoCl 2(mM)015050100140101491150
recombinant BMP2protein containing the poly-His tag was expressed and puri?ed using a Ni af?nity column under denaturing conditions according to the manufacturer’s protocol(Invitrogen).
The BMP2was added directly within the core part of the core–shell scaffolds during the preparation of scaffolds.BMP2solution was mixed with the collagen solution which was then produced into core–shell scaffolds.The crosslinking solution was?xed at 149mM CaCl2and1mM CoCl2.For the release study of BMP2,each scaffold was placed in each well of24-well plates containing1.5ml of distilled water.After incubation for different durations,the med-ium was replenished,and the supernatant was analyzed by enzyme linked immunosorbent assay(ELISA,Perrotech).
2.3.Rat mesenchymal stem cells and cell viability on scaffolds
Mesenchymal stem cells(MSCs)derived from rat bone marrow were harvested from the femora and tibiae of adult rats(180–200g)according to the guidelines approved by the Animal Ethics Committee of Dankook University.The harvested product was then centrifuged,and the supernatant was collected and suspended within a culture?ask containing a normal culture medium (a-minimal essential medium(a-MEM)supplemented with10% fetal bovine serum(FBS),100U/ml penicillin,and100mg/ml streptomycin),in a humidi?ed atmosphere of5%CO2in air at 37°C.After incubation for1day,the medium was refreshed and cultured until the cells reached near con?uence.After the subcul-ture was maintained in the normal culture condition,cells at2–3 passages were used for further tests.
The effects of Co ions incorporated within the scaffolds on the cell viability were?rst assessed.An indirect culture method was used.Cells were seeded at10,000on each well of24-well plates containing1.5ml of normal culture medium.After24h,the scaf-folds were placed on top with the help of Transwell inserts.The scaffolds analyzed were those crosslinked in150mM CaCl2, 149mM CaCl2+1mM CoCl2,and140mM CaCl2+10mM CoCl2. The tissue culture plate without any scaffolds was used as a con-trol.Cell proliferation was measured after1,3,and6days using a cell counting kit-8reagent(CCK-8,Dojindo Molecular Technologies).To perform the CCK-8assay,the medium was removed and replaced with200l l of serum-free medium followed by the addition of20l l of CCK-8solution.The reagent was left to react for2h and the product obtained was then read at an absor-bance of450nm using a spectrophotometer.
For the optical microscopy of cell growth behaviors,at each cul-ture period,the cells were?xed with4%paraformaldehyde solu-tion,stained with Alexa Fluor546conjugated Phalloidin (Molecular Probes)and Prolong Gold antifade reagent with DAPI (Molecular Probes),and then visualized under?uorescence signals using a confocal laser scanning microscope(CLSM;LSM510,Zeiss).
2.4.In vitro angiogenesis study
The in vitro performance of the designed scaffolds was?rst ana-lyzed based on the effects on cellular angiogenesis.The experiment was carried out using an indirect culture method with Transwell inserts,as described above.Four different scaffolds were used for the assays:incorporation of only Co,of only BMP2,of both Co and BMP2,and with no incorporation.The scaffolds that included Co ions were crosslinked within149mM CaCl2+1mM CoCl2, and those that incorporated only BMP2were crosslinked in 150mM CaCl2.Cells cultured without scaffolds were used as a con-trol.Culture periods were up to6days.
2.4.1.Expression of angiogenic genes
The expression of genes associated with angiogenesis was assessed by quantitative PCR(qPCR).After3and6days of culture,each sample was washed with PBS twice and the cells were detached by trypsinization.The cell pellet was collected and frozen at-80°C until use.Total RNA was extracted from the cells using the RNeasy Kit(Qiagen)according to the manufacturer’s instructions. The concentration of RNA samples was quanti?ed using a Nanodrop2000spectrophotometer(Thermo Scienti?c).RNA sam-ples were transcribed into cDNA by combining the RNA with a ran-dom hexamer,followed by denaturation at70°C for5min.After denaturation,the samples were mixed with the RT premix for complete cDNA synthesis.cDNA was then ampli?ed using RT-PCR premix(Bioneer)at94°C for5min,followed by35cycles of94°C for1min denaturation,57°C for1min for annealing,and 72°C for1min for extension.Primers for ampli?cation were CD31: CTCCTAAGAGCAAAGAGCAACTTC(forward),TACACTGGTATTCCAT GTCTCTGG(reverse),VEGF:ACCAGTGACAGAGGCCAATACT(for-ward),GGCCTCCACAGTCAGGTTATAC(reverse),HIF-1a:GCGTACA TAGGGACTGGAGA(forward),AAAGTGGGTAGGAGAAAGGG (reverse)and glyceraldehyde-3-phosphate dehydrogenase (GAPDH):TGAACGGGAAGCTCACTGG(forward),TCCACCACCCTGT TGCTGTA(reverse).The results were normalized with respect to GAPDH.
2.4.2.VEGF production
To determine the ability of the cells to secrete angiogenic fac-tors,the release of VEGF was quanti?ed by ELISA(ELISA, Perrotech).After culturing,the medium was collected every two days and stored at-20°C until further analysis.After6days,an ali-quot of each sample was used to quantify the amount of VEGF pro-duced.The results were normalized to the cell number.
2.4.
3.Matrigel assay for tubular formation
In vitro tubular formation was examined using a Matrigel(BD Biosciences,BD Matrigel,356231)culture.The Matrigel was coated onto24well plates for1h at37°C for complete gelation,according to the manufacturer’s speci?cations.80,000cells were then cul-tured on each well plate and Transwell inserts containing different scaffolds were placed on top.The cells were monitored by optical microscopy at3,6,and22h of culture.The images were then ana-lyzed with Metamorph software in order to measure the length of the tubules formed.
2.5.In vitro osteogenesis study
In order to examine the ability of the scaffolds to induce osteo-genic differentiation of rMSCs,a similar experimental design was used as that described above,involving four different scaffold groups and a culture dish control,and the Transwell inserts culture method was used.Culture periods were prolonged up to7and 14days.
2.5.1.Expression of osteogenic genes
The expression of genes associated with osteogenesis,including osteocalcin(OCN),alkaline phosphatase(ALP),bone sialoprotein (BSP),and osteopontin(OPN),was assessed by q PCR.After culture for7and14days,the cells were trypsinized and the cell pellet was collected and frozen at-80°C until used.The total RNA was extracted from differentiated cells using the RNeasy Kit(Qiagen) according to the manufacturer’s instructions.The concentration of RNA samples was quanti?ed using a Nanodrop2000spectropho-tometer(Thermo Scienti?c).The RNA samples were transcribed into cDNA by combining the RNA with a random hexamer,fol-lowed by denaturation at70°C for5min.After denaturation,the samples were mixed with the RT premix for complete cDNA syn-thesis.cDNA was then ampli?ed using RT-PCR premix(Bioneer) at94°C for5min,followed by35cycles of94°C for1min denat-uration,57°C for1min for annealing and72°C for1min for
298R.A.Perez et al./Acta Biomaterialia23(2015)295–308
extension.The primers used for ampli?cation were OCN: CATGAAGGCTTTGTCAGACT(forward),CTCTCTCTGCTCACTCTGCT (reverse);ALP:CCTTTGTGGCTCTCTCCAAG(forward),CGATGTCCT TGATGTTGTGC(reverse)BSP:AGAAAGAGCAGCACGGTTGA(for-ward),TCATAGCCATGCCCCTTGTA(reverse);OPN:GAGGAGAAG GCGCATTACAG(forward),AAACGTCTGCTTGTCTGCTG(reverse); and glyceraldehyde-3-phosphate dehydrogenase(GAPDH): TGAACGGGAAGCTCACTGG(forward),TCCACCACCCTGTTGCTGTA (reverse).Results were normalized to GAPDH.
2.5.2.OCN immuno?uorescence staining
As the representative osteogenic marker at a relatively later stage,OCN protein expression was examined using immuno?uo-rescence staining.After7and14days,the cells grown on each sample were?xed with4%paraformaldehyde and then treated with0.1%Triton X-100.After washing with PBS,the samples were immunostained against OCN antibody to observe osteogenic mar-ker OCN.Alexa Fluor546-conjugated phalloidin(Invitrogen A22283)diluted in PBS was also added to each sample to stain the F-actin.A Prolong Gold antifade reagent with4’,6-diamidi no-2-phenylindole(DAPI;Invitrogen)was used to stain the cells and to produce blue?uorescence signals upon binding to the DNA in the nucleus.Images were visualized under a Zeiss LSM 510microscope.
2.6.In vivo bone formation study
The bone forming ability of the scaffolds was examined in a rat calvarium defect model.Ten week old male Sprague–Dawley rats were used in the experiment.Animals were maintained in a12-h light/dark cycle(lights on from8:00to20:00),in a relative humid-ity(30–70%)and temperature(20–24°C)controlled environment. The rats were allowed a normal diet and water ad libitum.The experimental protocol for all animal care and use in this study was reviewed and approved in accordance with guidelines estab-lished by the Animal Care and Use Committee,Dankook University,Cheonan,Korea.
2.6.1.Animal surgery and scaffold implantation
The animals were anaesthetized by intramuscular injection with a mixture of ketamine(80mg/kg)and xylazine(10mg/kg) for surgery.The hair over the skin on the dorsal region of the cra-nium was shaved and the region was aseptically prepared using povidone and70%ethanol for surgery.A linear sagittal midline skin incision was made over the calvarium with a#10surgical blade.A full-thickness?ap of the skin and periosteum was elevated to obtain a suf?cient surgical?eld for the drilling.Two 5mm-diameter critical sized circular full-thickness bone defects were created at the center of each parietal bone in the calvarium of each rat.To create the defects without overheating of the bone edges,a trephine bur was used under constant irrigation with nor-mal saline solution,without damaging the underlying sagittal sinus and dura matter.The scaffolds were sterilized prior to use. Each cranial defect was randomly?lled with0.1ml with the four different groups:with no Co/BMP2,Co only,BMP2only,and Co/BMP2both,having two different defects per animal.In all rats, subcutaneous tissue was closed with absorbable materials,and the skin incisions were sutured with non-absorbable materials.
After the implantation,the animals were housed individually in cages and monitored by visual observation during the experimen-tal time for signs of infection,in?ammation,and any adverse reac-tion.Six weeks after the operation,the animals were sacri?ced to collect the calvariae.The skin was dissected,and the tissues includ-ing the surgical sites and their surrounding tissues were harvested along with the surrounding bone.The specimens were?xed in10%buffered neutralized formalin for24h at room temperature and prepared for micro-CT(l CT)analysis and histology.
2.6.2.Micro-computed tomography analysis
Following?xation,the micro-computed tomography(l CT) images were performed to visualize the samples and to analyze images of new bone formation.The harvested specimens were scanned using a l CT scanner(Skyscan1176;Skyscan,Aartselaar, Belgium)with a camera pixel size of12.56l m;also,a frame aver-aging of3was employed together with a?lter of1mm aluminum, a rotation step of0.5°,and a rotation angle of180°.The X-ray tube voltage was65kV and the current was385l A,with an exposure time of279ms.A cylindrical region of interest(ROI)was posi-tioned over the center of the single defect,fully enclosing the new bone within the defect site.The percentages of new bone vol-ume(%),bone surface area(mm2),and bone surface density (1/mm)of newly formed bone within each ROI were measured by assigning a threshold for the total bone content(including tra-becular and cortical bone)through a computer analysis program, CTAn(Skyscan).
2.6.
3.Histological procedure
After l CT imaging,the harvested samples were prepared for histological analysis.To achieve this,?xed specimens were decalci?ed with RapidCal TM solution(BBC Chemical Co., Stanwood,WA,USA),dehydrated through a series of ethanol solu-tions of increasing concentration(from70%to100%),and subsequently embedded in paraf?n.Five micrometer thick coronal sections were prepared at the central region of the circular defects and de?ned as the representative histology.The slides were stained with hematoxylin&eosin(H&E)stain using a routine tech-nique and examined using a light microscope(IX71,Olympus, Tokyo,Japan).
2.7.Statistical analysis
Experiments were performed in triplicate,unless otherwise speci?ed.Data are expressed as mean±one standard deviation. Statistical analysis was carried out using one way analysis of vari-ance(ANOVA)with a Fisher post hoc test and a signi?cance level was considered at p<0.05.
3.Results
3.1.Core–shell designed scaffolds
The images of the core–shell?brous scaffolds produced are shown in Fig.1b.The optical image shows the transparent?brous structured scaffolds in the medium and the enlarged image shows the core and shell part of the?ber with an average core diameter of $1000l m and an average shell thickness of$150l m.The?brous morphology of the scaffolds was well preserved in the culture medium and easily formed into the shape of a mold.The SEM mor-phologies of the freeze-dried scaffolds exhibited a highly macrop-orous structure and the core–shelled structure can be seen in cross-section.No difference was observed in the morphology of the core–shell?brous scaffolds depending on the Co/Ca composi-tions used for the alginate crosslinking.
The release of therapeutic molecules,including Co in the shell and BMP2in the core,was monitored as shown in Fig.2. Scaffolds were prepared with different crosslinking solutions while varying the concentrations of Co ions from0to150mM which was balanced with Ca ions.The Co and Ca ions will be competitively incorporated into the alginate shell part during the crosslinking, and the incorporated amount of Co could also be assessed
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indirectly by the complete release amount of Co.The results showed that when the scaffolds were crosslinked in 150mM Co solution,the Co release amount increased gradually with time,showing almost 6mM for 6days (Fig.2a).When 100mM Co was used,approximately 2mM Co was released for 6days.The release pro?les were similarly observed in the samples when lower Co contents were used for crosslinking (10mM and 1mM Co in the enlarged graphs).As a consequence,the Co release was 6mM,2mM,0.15mM,and 0.015mM,respectively,when the Co concen-tration used was 150mM,100mM,10mM,and 1mM,as plotted in Fig.2b.The log–log plot ?tted well from a linear regression (with R 2=0.989),suggesting that Co incorporation is tunable by chang-ing the Co content of the crosslinking solution.The Co incorpora-tion and release amount should be critically considered when utilizing the Co-incorporating scaffolds for the purpose of angio-genic scaffolds while avoiding toxic effects on cells.Moreover,the Co release was relatively rapid,exhibiting an almost complete release within a week.
The BMP2release from the core part of the scaffolds was also measured using ELISA,and the amount released was assessed rel-ative to the initial loading quantity (Fig.2c).The release pro?le was fairly gradual,with a smooth curved pattern over the recorded per-iod of 21days,without showing any initial burst effect.The amount of BMP2released for 21days was only 16%,implying still a large amount of BMP would be remained.The BMP2release was more sustained than the Co release.The combined release graph of BMP2with Co (1mM represented)shows clearly the different releasing pro?les (Fig.2d).While the Co was released rapidly within a week,the BMP2release was continued over several weeks,con?rming the time-dependent sequential release behaviors of the two therapeutic molecules.
Using the therapeutically designed scaffolds that are to carry both Co and BMP2,we ?rst assessed cell viability of MSCs,partic-ularly as a function of Co concentration.MSC viability was assessed in indirect contact with the scaffolds crosslinked with different Co concentrations (0,1mM and 10mM with Ca ion balanced to 150mM)at the culture periods of 1,3,and 6days.The cell count-ing kit assay showed that increasing Co concentration decreased cell viability (Fig.3a).While there was little cell proliferation over time when 10mM Co was used,the cells appeared to proliferate actively when the Co used was at a relatively low level (1mM).The ?uorescence images of cells clearly re?ected the cell viability result (Fig.3b).Based on this,the scaffolds crosslinked with Co at a concentration of 1mM were used for further experiments.3.2.In vitro angiogenesis behaviors
The effects of the scaffolds on angiogenesic responses of MSCs were examined.The expression of angiogenic genes,including CD31,VEGF,and HIF-1a ,were quanti?ed by RT-PCR,as shown Fig.4.The pro-angiogenic stimulation by the Co ions was clearly observed particularly in the early culture period;on the other hand,the use of BMP2was not effective in increasing angiogenic gene expression.
We further measured VEGF protein release from MSCs in response to the different therapeutic scaffolds over the culture per-iod of 6days,as shown in Fig.5.The cumulative release was assessed using an ELISA kit and then normalized to cell number.A signi?cantly higher amount of VEGF was secreted by cells in the scaffolds that incorporated Co ions,and the up-regulation was about 1.5–2times greater than those scaffolds without Co.BMP2did not affect VEGF
secretion.
2.Release pro?les of Co and BMP2from the therapeutically-designed core–shell scaffolds.(a)Co ionic release (l M)dependent on the concentration of CoCl 2(0,1,10,100150mM)used initially for the alginate crosslinking solution.Three different graphs plotted to better present different Co concentration regions (note a signi?cant difference in y-scale).Based on the Co release,the incorporation of Co was calculated to be $150l M and 15l M for the 10mM and 1mM Co crosslinking solution,respectively.Co release rapidly occurs for all cases,reaching a plateau as early as within a weeks.(b)Co ion incorporated platted with respect to the Co ion initially used in crosslinking solution.The Log–log plot shows a linear relationship with R 2=0.989.(c)BMP2release from core–shell scaffolds crosslinked in 1mM CoCl 2(149mM CaCl 2),measured by ELISA,showing a gradual long-term release pro?le.Only 16%of BMP2released after 21days,suggesting an on-going release possibly over a month.(d)Both BMP2and Co release (1mM Co)co-plotted to show their time-dependent sequential release behaviors.
The ability of therapeutic core–shell scaffolds to promote for-mation of tubule-like structures was further assessed by indirectly culturing MSCs on Matrigel substrate,which was affected by the Co ions and BMP2released from the scaffolds.After culturing for dif-ferent time periods,cellular morphology and formation of tubules were assessed(Fig.6a–c).Cells initially(at3h,Fig.6a)appeared to be randomly distributed,but subsequently rearranged and aligned to form tubule-like structures(at6h,Fig.6b),?nally forming thickened and continuous tubular networks(at22h,Fig.6c).In particular,this cellular rearrangement into a tubular network was more apparent in the Co-incorporating scaffolds.Without Co, many areas were merely cellular aggregates without branched, tubular networks(highlighted in Fig.6c).From a quanti?cation of the tubular length,signi?cantly higher values were observed on the Co-incorporated scaffolds(Fig.6d).3.3.In vitro osteogenesis behaviors
After the analysis of the effects of the Co-and BMP2-delivering scaffolds on angiogenic stimulation,their ability to promote osteo-genesis was assessed.The expression of osteogenic genes including OPN,OCN,BSP,and ALP was quanti?ed by RT-PCR analysis,as shown in Fig.7.The group-dependent expression patterns were similar for all four genes.At7days,all the therapeutic scaffolds that incorporated Co and/or BMP2showed signi?cantly higher gene expression levels than the bare scaffolds.Interestingly,the scaffolds incorporating Co ions either with or without BMP2, upregulated the gene levels to a higher degree;however,at a pro-longed period(14days),those incorporating both Co and BMP2 showed the highest level.Results demonstrated the effects of both
of Co concentration incorporated within the core–shell scaffolds on MSC viability.(a)Cell proliferation measured in indirect contact with
different Co concentrations(0mM,1mM and10mM Co)after1,3,and6days using CCK-8reagent.Statistically signi?cant differences are letters(p<0.05).(b)Phalloidin(green)and DAPI(blue)staining of the MSCs in contact with different cobalt concentrations.Scale bar=350l m.
4.Effects of different therapeutic designs of the core–shell scaffolds loading Co and/or BMP2on the angiogenesis of rMSCs.Expressions of angiogenic genes,including CD31,VEGF,and HIF-1a,were analyzed by quantitative PCR.
Fig.5.VEGF protein release secreted by the rMSCs affected by differently-designed
therapeutic scaffolds during a6days culture.Cumulative release assessed by an
ELISA kit,was normalized to the cell number.Statistically signi?cant differences are
indicated with different letters(p<0.05).
Co and BMP2on the stimulation of a series of osteogenic genes in
MSCs during the indirect culture with the therapeutic scaffolds.
To further con?rm the osteogenic stimulation,the OCN,a mar-
ker engaged at a relatively late period of osteogenesis,was
immuno?uorescence-stained at14days of cell culture,as shown
in Fig.8.Nuclei and cytoskeletons were co-stained with DAPI
(blue)and phalloidin(red),respectively.Green OCN signals were
the most apparent in the group with Co and BMP2when compared
to other signals.
the therapeutic core–shell scaffolds to promote the tubular formation of MSCs seeded on the Matrigel model substrate after(a)3h,(b)
tubular length.Statistically signi?cant differences were noticed between groups considered at each time point(?p<0.05).Scale bar in
appeared to be similar for all scaffold groups.However,a higher magni?cation of histologic images showed more clearly that the neo-tissues formed around the scaffold as well as at the interface of scaffold/neo-tissues (Fig.10b).All the scaffolds appeared to indi-cate partial in vivo degradation.On closer examination,the neo-tissues in the bare scaffold (without Co and BMP2)were com-posed primarily of ?brous tissues with blood vessels and only a small fraction of the areas indicated calci?ed tissue.However,in the Co/BMP2co-delivered scaffold,the surrounding tissues were highly calci?ed,showing a bone-like structure,and this calci?ed tissue comprised most of the neo-formed tissues.Osteoblasts actively depositing ECM and mineral on the surface of the scaffold were observed throughout the newly formed bone.Some blood vessels were also clearly seen.This calci?ed tissue type was found to be more conspicuous in the BMP2-releasing scaffolds,most notably in the Co/BMP2co-delivering scaffold,coinciding with the l CT analyses.The l CT and histological results together gave the evidence that the bone-like calci?ed tissues were formed sur-rounding the scaffolds.4.Discussion
The development of therapeutic scaffolds with the capacity to deliver drugs and biomolecules in a controllable and sustained manner constitutes one of the most promising ?elds in bone regen-eration.Among the strategies,multiple drug delivery systems with a sequential release pro?le are thought to effectively repair and regenerate tissues including bone.In the bone repair process,rela-tively early therapeutic events including anti-in?ammation,angio-genesis,and stem cell homing ability,help the accompanying target actions of bone cell differentiation and calci?cation [20].The most widely-studied therapeutic combination,among other drugs and growth factors,is VEGF with BMP2,to improve and syn-ergize angiogenesis and osteogenesis [21,22].Moreover,speci?c scaffold designs and compositions have been sought to incorporate molecules safely in large quantities and to deliver them timely and sequentially [5,6].
In this study,we report for the ?rst time the combination of Co ions with BMP2,anchored within a core–shell scaffold,with the
objective of enabling sequential delivery of both therapeutic mole-cules to ultimately realize effective bone regeneration.The core–shell design enables unique features of scaffolds for use as drug delivery vehicles.The therapeutic molecules and ions can be encapsulated and partitioned within the layered core–shell struc-ture,and then released to the site of defect in a diffusion depen-dent manner,even facilitating sequential delivery of multiple molecules.Furthermore,the release pro?les can be easily tuned by changing the composition and size of the core and shell part [23].In particular,the use of Co ions as the pro-angiogenic factor has rarely been exploited within scaffolding systems.In fact,Co has been shown to mimic a certain level of hypoxic conditions,which are known to induce angiogenesis [10–12].Furthermore,Co has also been shown to be an effective ion that might stimulate osteogenesis [24,25].A major rationale for using Co ions is related to our scaffolding system,the collagen core with alginate shell,where the alginate is designed to crosslink with the help of Co ions while simultaneously incorporating them within the structure.Along with Co ions,the BMP2protein is easily loaded on demand onto the core–shell scaffolds.In particular,altering the Co concen-tration in the crosslinking solution resulted in tunable amounts of Co ions being loaded.As the Co loading occurs by a diffusion pro-cess through the alginate network,the initial solution concentra-tion determines the driving force for ionic diffusion.As the Co ions were initially balanced with Ca ions in the solution,the Ca ions also diffuse to alginate and thus devote in part to the crosslink.Therefore,the subsequent Co ion release should be in?uenced both by the amount of Co ions incorporated and by the alginate cross-link state.
The core–shell hydrogel design has proven to be effective in releasing incorporated molecules in a sequential manner.Co ions were released mostly within 7days,while BMP2release continued over a longer period of at least several weeks,with the possibility of sustained release over months based on the released percentage during the test period.As deduced from the alginate crosslinking mechanism,the Co ions are considered to be present primarily within the alginate network in the shell;however,a certain amount should also be interactive with the a -TCP powders added at 10%.As demonstrated in our previous work,the negative-charged
surface
of the therapeutic scaffolds on the osteogenesis of rMSCs.Expression of osteogenic related genes,including OCN,OPN,BSP,and ALP,was PCR at 7and 14days.Statistically signi?cant differences are indicated with different letters (p <0.05).
of the powder will interact with the positive-charged molecules,improving the binding ability,and thus sustaining the release from a few days to a week [8].Although this is more effective in the case of positive-charged proteins,some effects might also be envisaged in the cations.BMP2had a more sustained release pro?le.In fact,the collagen molecules placed in the core part are known to easily bind with growth factors including BMPs,while preserving the pro-teins’structure.Our previous works on the release of other growth factors,such as bFGF and NGF,from the collagen gel matrix have also demonstrated quite sustainable release over two weeks [26,27].In addition,the shell layer might also delay the diffusion of the BMP2molecules.The a -TCP incorporated as high as 50%should also facilitate sustaining the release of BMP2.As a conse-quence,the BMP2release over at least 3–4weeks was achieved,and possibly even months,which is considered to be highly bene?-cial for in vivo bone regeneration [5].
The cellular toxicity ascribed to the Co ions released from the scaffolds is a primary index for the biological usefulness the novel therapeutic-designed scaffolds.In fact,studies have used Co ions (CoCl 2)to simulate hypoxic cell culture conditions,where the common concentration used was 100l M [15,16,28].Taking the release amount of Co ions into consideration from our results (Fig.2),10mM of crosslinking solution was ?rst considered,but this was proven to reduce the proliferation capacity of rMSCs.Rather,a much lower concentration of Co (1mM solution,an order of magnitude down),which corresponded to Co release amount of $10l M,was shown to be cell viable,with an on-going rMSCs’proliferation over the test periods (Fig.3).Several studies have also reported the Co ion-induced suppression of cellular growth and the signi?cant dose-dependent toxicity behaviors (1to 100l M Co ions)[16,29].While suppressing a cer-tain level of cellular viability,the Co has also been shown to sig-ni?cantly improve the angiogenesis,including VEGF production through the HIF-1a pathway [29].However,most of the works on hypoxic mimic cell cultures using Co ions have introduced cell lines for cancer study;therefore,the optimal conditions (particu-larly in terms of the Co concentrations that are not toxic but induce angiogenesis)will differ from our study,since we used dif-ferent cell type and strategized a scaffold-based Co release sys-tem.Taken together,the scaffolds crosslinked in 1mM of Co solution were considered for further in vitro angiogenesis and osteogenesis studies using
rMSCs.
images of OCN immunostaining after 14days of MSCs culture in indirect contact with the core–shell scaffolds,with the nucleus stained in cytoskeleton in red (phalloidin).Scale bar =100l m.OCN green signals were revealed apparently only in BMP2(+)groups.
The Co-delivering scaffolds clearly supported stimulation of angiogenic responses,characterized by the expression of a series of angiogenic genes,production of VEGF,and formation of cellular tubule-like networks (Figs.4–6).The signi?cant up-regulation of genes,including HIF-1a ,CD31,and VEGF,particularly at the early culture period in the Co-releasing scaffold,is a strong sign of the MSCs’capacity to provide vascular support [30].HIF-1a ,character-istically up-regulated in hypoxic environments and by Co ions,is known to stimulate other angiogenic factors such as VEGF [10,11].The transient stimulation of those angiogenic genes,within short time frames ($a few days)observed in this study,has also been reported for endothelial cells and other stem cells that were committed to functional differentiation within short time periods in vitro [31,32].Along with this change in mRNA level,the expression of protein was also assessed.VEGF was produced at greater levels by MSCs on the Co-containing scaffold.While the MSCs secreted a substantial level of VEGF protein even on the Matrigel culture,the Co ions acted as a soluble inducer and syner-gized with Matrigel substrate to enhance VEGF secretion.
MSCs have been shown to assemble into tubule-like structures when cultured on Matrigel.However,mesenchymal cell character-istics are maintaining with the cells typically positive for smooth muscle cell markers,but rarely positive for endothelial markers [33].Similarly in this study,the fate of MSCs is not thought to be endothelial,rather a vascular support phenotype that can form tubule-like structures.While the Matrigel provides mainly physical and adherent chemical cues to MSCs,the Co ions released from the core–shell scaffolds should act as a soluble biochemical signal to drive differentiation into cells that support vascular networks.So,while MSCs will not form functional blood vessels per se ,they will probably exhibit enhanced angiogenic behavior in vivo due to Co release from our scaffolds.Therefore,the consistent in vitro results demonstrating the stimulatory role of Co on MSCs allow some use-ful insight on the possible pro-angiogenic ef?cacy of our scaffolds.In some recent works,incorporating Co ions in scaffolds has also been shown to improve the VEGF production and angiogenic func-tions of MSCs in vitro [12,25].In one study,the Co ions released have also stimulated in vitro osteogenic differentiation,although the effects were counterbalanced with lowered cell viability,implying the usefulness of the Co-delivering scaffolds for improv-ing bone repair.
Together with the angiogenic effects,the Co/BMP2delivery sys-tem has demonstrated enhanced osteogenic differentiation of MSCs in vitro .A series of osteogenic genes,including OPN,ALP,BSP,and OCN,were identi?ed to be up-regulated by the scaffolds,delivering either Co or BMP2or both.Interestingly,the highest up-regulation was noticed on those scaffolds that delivered only Co ions for the period of 7days.The release of BMP2was not syn-ergistic at that time,but at a much later time of 14days,with the Co/BMP2co-delivered group showing the highest gene
expression.
bone forming ability of the therapeutic-designed core–shell scaffolds after 6weeks implanted in a 5mm defect of rat calvarium.Bone regeneration microcomputed tomography (l CT);(a)l CT constructed images showing the new bone formation (white color)around the 5mm diameter CT analyses showing (b)percent of new bone volume,(c)bone surface area,and (d)bone surface density.Signi?cant differences are indicated 0.05).
Although the bene?t of either Co or BMP2on osteogenic gene expression was apparent for all genes with respect to the non-delivered scaffold,the time-dependent up-and down-regulations and the merely synergistic role of BMP2have yet to be clari?ed in subsequent studies.However,in the current study,characteriza-tion of OCN protein immunostaining at day 14demonstrated the stimulatory roles of BMP2.The transient-expressing nature of genes and the gene levels that do not match directly with the sub-sequent protein syntheses might complicate the in vitro results.Based on the in vitro studies on rMSCs,Co ions were demon-strated to play signi?cant roles in pro-angiogenic and osteogenic differentiation.Relative to Co ions,BMP2,known as one of the most potent osteogenic inducers,showed some effects in osteoge-nesis whilst little function in angiogenesis.In fact,in terms of the Co ions,many in vitro studies have also demonstrated osteogenic stimulatory roles,in enhancing expression of osteogenic genes through the signaling by angiogenic factors such as VEGF [34–37].Previous work has also treated bone marrow stromal cells with Co ions for the regeneration of the periosteum,and showed that the in vitro Co treatment increased VEGF gene expression,which in turn enhanced vascularization and osteogenesis in vivo [15].Therefore,the use of Co ions is considered a rational strategy to stimulate osteogenesis in vitro for subsequent bone tissue engi-neering.Our in vitro results revealed that Co ions had substantial stimulatory effects on angiogenesis and to some degree on osteo-genesis,while the BMP2functioned less profoundly in osteogene-sis without synergizing the Co function on MSC behavior.However,while many studies have proven the ability of BMP2in in vivo bone formation,the in vivo roles should not be underestimated.
The in vivo performance of the Co/BMP2-delivering therapeutic scaffolds was investigated in a rat calvarium model.The results showed the effective roles of BMP2in bone formation,on the basis of l -CT analyses and histological evaluations (Figs.8and 9).The BMP2-delivered scaffolds,either with or without Co,improved all
the bone formation indices analyzed by l -CT,including bone vol-ume percentage,and the bone surface area and density,up to approximately twice that of the control scaffolds in which these factors are absent.On the other hand,the role of Co,which had sig-ni?cant osteogenic effects on rMSCs in vitro ,has rarely been inter-rogated in vivo .In other words,the involvement of BMP2has proven to be critical in induction of bone formation in vivo around the implanted scaffolds.Apart from the innate difference between the physiological ?uid and in vitro culture medium conditions,the environments experienced by the cells surrounding the implanted scaffolds in vivo will differ from those in the in vitro cultures.While we assessed the MSCs’behavior indirectly (using Transwell inserts where they would be affected solely by the soluble factors (Co ions and BMP2)),in vivo bone formation was the result of the cellular responses with the scaffolds.At this point,we observed from the histologic images that the scaffold-occupying area (from 2D pro-jections)is as high as $60–70%of the total defect volume,due to the high packing density of scaffolds,which may reduce pore avail-ability for vascularization,possibly being simulated by the Co ions.Therefore,a less packed scaffold to enable a larger pore volume might be preferred to examine the cellular ingrowth and vascular-ization in vivo ,which needs future study.
Another aspect to consider in the in vivo design is that the scaf-folds showed very limited sign of degradation;rather,most of the scaffolds appeared to remain during the implantation period of 6weeks.This was probably due to the use of a -TCP self-hardened powder,which initially was incorporated to improve the stiffness of scaffolds from tens of kPa to hundreds of kPa [8],better mimicking the native bone matrix,and thus favoring osteo-genesis.The continuous release of Ca ions from the transformed hydroxyapatite with time might have a signi?cant effect on the alginate degradation.Alginate cross-links in the presence of diva-lent ions,whereas it easily degrades in the presence of monovalent ions.The presence of the bivalent Ca ions allows the
continuous
10.Representative histological images of the implanted samples for 6weeks,following hematoxylin and eosin (H&E)staining at low (a)and high magni?cation (b).1mm.
displacement of the possible monovalent ions found in physiolog-ical?uids,stabilizing the alginate network.On the other hand,the sustained release of BMP may also indicate reduced biodegradabil-ity.Since we have already observed the rapid degradation of collagen-alginate core–shell hydrogels both in vitro and in vivo [9],the slow degradation of the current hydrogels in vivo was pri-marily deemed to be due to the presence of a-TCP,as it increases the stability of alginate and collagen and it has its own low degrad-ability.In fact,the neo-bone volume of the scaffolds ranged at a relatively low level,approximately15–35%,even though the l CT projection images showed substantial?lling of defect regions.On the other hand,the bone surface area was as high as50–100mm2,values which are generally recorded in high bone vol-umes(mostly over50%bone volume).The discrepancy between the bone volume and surface indices was reasoned to be that because the neo-bone formation could only occur within the lim-ited volume through the scaffold surface and within interspacings, while scaffolds mostly remained without being replaced by neo-bone tissues.This was more clearly observed in the histologi-cal?ndings.
However,in terms of the quality of neo-bone tissue formed in the pore spaces,the delivered molecules enhanced this.In the BMP2and Co/BMP2delivery scaffolds,the newly-calci?ed tissue outlined the scaffold uniformly and thoroughly,and?lled the pore spaces profoundly;however,in the scaffolds without the delivery of Co and BMP2,most of the pore regions showed?brous tissue formation with only minimal signs of calci?ed matrices.When Co ions were co-delivered with BMP2,signi?cant synergistic effects were observed on both the bone volume and surface, although the difference was not as signi?cant as the action of BMP2.Previous results have also demonstrated that the use of angiogenic factor VEGF together with BMP2could not effectively synergize the roles of BMP2in bone formation[5,6].Although the therapeutic scaffolds remained largely intact,with limited bone regeneration in terms of bone volume,the bone surface qual-ity was excellent,possibly forming an interface well-integrated with the scaffolds.Furthermore,the scaffolds had stiffness levels that matched the bone matrix fairly well,with excellent tissue compatibility;therefore,the hydrogel-like scaffolds forming an interwoven structure with the newly-formed bone matrix are con-sidered to function properly,awaiting a long-term degradation and a slow replacement by neo-bone tissues.This study demonstrates the in vitro and in vivo biological roles of the newly designed core–shell hydrogel scaffolds to deliver Co ions and BMP2in a sequential manner,ultimately suggesting potential therapeutic scaffolds for bone regeneration.
5.Conclusions
Novel collagen/alginate-based core–shell hydrogel scaffolds were designed to deliver Co ions and BMP2in a sequential manner for synergizing angiogenic and osteogenic events,effective for bone regeneration.The scaffold process enabled simple and tun-able incorporations of the Co ions and BMP2within each compart-ment;furthermore,their release was highly sequential,with a rapid Co ion release within a week and a highly sustained BMP2 release over several weeks to months.In vitro studies performed with rMSCs demonstrated the roles of Co ions in angiogenesis at the early culture period as well as the osteogenic potential of BMP2at a much later period.In vivo rat calvarium tests of the scaf-folds con?rmed the synergistic roles of BMP2/Co in improving bone formation.While further advanced scaffold designs are still needed,this?rst report on utilizing Co ions with an osteoinductive factor is considered to facilitate a new class of therapeutic scaf-folds,aiming at successful bone regeneration with the help of angiogenesis.Acknowledgements
This work was supported by Priority Research Centers Program (Grant#:2009-0093829)through the National Research Foundation(NRF)funded by the Ministry of Education,Science and Technology,South Korea.
Appendix A.Figures with essential color discrimination Certain?gures in this article,particularly Figs.1–3,6,8and10 are dif?cult to interpret in black and white.The full color images can be found in the on-line version,at https://www.doczj.com/doc/fa11287507.html,/10.1016/ j.actbio.2015.06.002.
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１２Ｓａｎｃｈｅ｝Ｒ目１喵Ｊ．ＳｏｎｇＳ．Ｃａｒｄｅｚｏ－ＰｅｌａｅｚＦ．ｅｔａ１．Ａｄｕｌｔｂｏｎｅｎｍｒ— Ｗｗｓｔｒｄｎａｌｃｅｉｌｓｄｉｆｆｅｒｅｎｔｉａｔｅｉｎｔｏｎｅｕｒａ】ｃｅｌｌｓｉｎｖｉｔｒｏ［Ｊ］ＪＥｘｐＮｅｕｍｌ，２０００，１６４（２）：２４７～２５６ １３Ｄ眩自ｗａＭ．ＴａｋａｈａｓｈｉＩ，ＥｓａｋｉＭ．ｅｌ＂ｄＳｃｉａｔｉｃｎｅｌｗｅｄｒｅｇｅｎｅｒａｔｉｏｎｉｎｒａｔｓｉｎｄｕｅｅｄｂｙｔｒａｎ旦ｐｌｍａｔａｔｉｏｎｏｆｉｎｖｊｔｒｏｄｉｆｆｅｒｅａｔｔｉａｔｅｄｂｏｎｅｍａｒ－ｒｏｗＳｔｌ＂ｏｒ—ｃｅｌｌｓ［Ｊ］ＥｕｒＪＮｅｕｒｃ８ｃｉ．２００１．１４（１１）：１７７１－－１７７６１４ＢｉａａｃｏＰ，ＲｉｍｉｎｕｃｃｉＭ．Ｇｒｏｎｔｈｃ６Ｓ，ｅｔａｌＢｏｎｅｒ／１；ａｒｒｏｗｓｔｌｖｍａｌｓｔｅｍｃｅｉｌｓ：ｎａｔｔｔｒｃ，ｂｉｏｌｏｇｙ，ａｎｄｐｏｔｅｎｔｉａｌａｐｐｆｉｃａｔｉｏｎｓ［Ｊ］ＪＳｃｉ一∞，２００１，１９（３）：１８０～１９２ １５ＢｉａｅｋＩＢ．Ｗ∞ｄｂ哪ＤＡｄｕｌｔｒａｔａｎｄｈｔｗｎａｎｂｏｎｅｍａｍｗ８ｔｒｏｒｄ ｓｔ￡ａ＇ｎｃｅｌｌｓｄｉｆｆｅｒｅｎｔｉａｔｅｉｎｔｏｎｅｕｍａｍ［Ｊ］ＪＢｌｏｏｄｃｅｌ】ｓＭｏｌＤｉｓ，２００１。２７（３）：６３２４６３６ １６Ｋｒａｔｔｓｅ１３５；Ｐｌａｓｔｉｃｉｔｙｏｆｒｍｒｍｗ－ｄｅｒｉｖｅｄｓｔｅｍｃｅｉｌｓ［Ｊ］．ＪＧ朗ｅ１ｋ，２００２．９（１１）：７５４－－７５８ １７Ｉ）ｅｍｗａＭＣｅｎｔｒａｌａｒｄｐｅｒｉｐｈｅｒａｌｎｅ—ｅｒｅｇｅｎｅｘａｔｉｏｎｂｙｎａｎｓｐＩａｎｔａ—ｔｉｏｎｏｆＳｃｈｗａｎｎｃｅｌｌｓａｎｄ删ｆｆｅｒｅｎｔｉａｔｅｄｂｏｎｅＩｎａｍｗｓｔｒ【１『ｎａｌｃｅｌｌｓ［Ｊ］ＪＡｎａｔＳｃｈｕｍａｎｎｅｑｕａｔｉｏｎＳｃｉＩｎｔ，２００２，７７（１）：１２－－２５ １８ＮａｕｍａｎｎＡ．Ｄ口谢ｓＪ．８ｔａｕｄｅｎｍａｉｅｒＲ，吐正Ｍｅｓｅａｃｈｙｍａｌ８ｔ邮１ ｃｅｌｌｓ－－ａｎｅｗｐａｔｈｗａｙｆｏｒｔｉｓｓｕｅｅｎｇｌｎｅｅｎｎｇｉｎｒｅｏｏｎｓｍｌｃｔｉｖｅｓｕｒｇｅｒｙ［Ｊ】ＪＬａｒｙｎｇｏｒｈｉｎｎｏｔｏｌｏｇｉｅ，２００２，８１（７）：５２１－－５２７ １９ＭｉｍｕｒａＴ，【ｋｚａｗａＭ．ＫａｎｎｏＨ，ｅｔａｌＰｅｎｐｈｅｒａｌ ｒ№ｍ姆ｆ啪一ｔｋｍｂｙｔｒｍｍｐｉａｎｔａｔｉｏｎｏｆｂｏｎｅｍａｒｒｏｗｓｎ℃ｎ诅ｌｅｅＵ＜ｌｅｒｉｖｅｄＳｃｈｗａｎｎｃｅｌｌｓｉｎａｄｕｌｔｒａｔｓ［Ｊ］．ＪＮⅢ％口ｇ，２００４，１０１（５）：８０６－－８１２ ２０ＣｏｎｇｅｒＰＡ．Ｍｉｎｇ，ⅧＪＪＰｈｅｎｏｔｙｐｉｃａｌａｎｄｆｕｎｃｔｉｏｎａｌｐｒｏｐｅｒｔｉｅｓｏｆｈｔｍｍａｂｏｎｅ１Ｔｌａｒ／ｏｗｍ＆ｅｎｃｈｙｎｕｄｐｒｎｇｅｍｔｏｒｅｅｌｌ５［Ｊ］ＪｃｄｌＰｈｙｓｉ—０１．１９９９，１８１（１）：６７—７３ ＡｎｔｈｏｌｏｇｙｏｆＭｅｄｉｃ—ｉ—ｎ—ｅ—，Ａｐｒ．２００６，Ｖ０１．２５，Ｎｏ．２—２１ＥｇｌｉｔｉｓＭＡ，ＭｅｚｅｙＥＨｅｍａｔｏｐｏｉｅｔｌｓｃｅｌｌｓｄｉｆｆｅｒｅｎｔｉａｔｅｉｎｔｏｂｏｔｈｍｉ— ｃｒｏｇｌｉａａｎｄｍａｃｍｇｌｉａｉｎｔｈｅｂｒａｉｎｓｏｆａｄｕｌｔｍｉｃｅ［Ｊ］ＪＰｒｏｃＮａｔｌＡ－ｃａｄＳｃｉＵＳＡ，１９９７，９４（８ｊ：４０８０－－４０８５ ２２ＣｕｅｖａｓＰ．ＣａｒｃａｌｌｅｒＦ．Ｄｕｊｏ，ｍｙＭ，ｅｔａ１．ＰｅｆｉｐｈｅｒＭｎｅｍｒｒｇｃｎｅｒａ－ｔｉｏｎｂｙｂｏｎｅｍａｒｒｏｗｓⅡ０ｒｒＩａｌｃａｌＬｑ［Ｊ］．ＪＮｅｕｒｏｋｗｉｃａｌＲｅｅｅａｒｃｈ，２００２，２４（７）：６３４－－６３５ ２３ＣｕｅｖａｓＰ．Ｃａｒｃｅｌ］ｅｒＦ，Ｇａｒｃｉａ．Ｇｏｒｎ觎Ｉ，ｅｔａｌＢｏｎｅｍａｒ工。ｗｓｔｒｃ前ｕｄｃｅｌｌｉｍｐｌａｎｔａｔｉｏｎｆｏｒｐｅｒｉｐｈｅｒａｌｎｅｒｖｅ地ｐａｉｒ［Ｊ］ＪＮｅｕｒａｌＲｅｓ．２００４，２６（２）：２３０—２３２ ２４ＫａｍａｄａＴ．ＫｎｄａＭ。Ｄ％ｍＭ。ｅｔａｌＴｒａｎｓｐｌａｎｔａｔｉｏｎｏｆｈｏｎｅｍａｒ－ｒｏｗｓｔｍｍａｌｃｄｌｄｅｒｉｖｅｄＳｏｈｗａｎｎ雌Ｕｓｐ工舢临ａｘｏｎｚｌｒｅｇｅｎｅｒａｔ；ｏｎａｎｄｆｕｎｃｔｉｏｎａｌｒｅｃｏｖｅｌＴｄｔｅｒｃｏｔｎｆＩｌｅｔｅｔｒａｎｓｅｃｔｉｏｎｏｆｅｄｕｌｘｍ∞ｉｎｄｃｏｒｄ［Ｊ］．ＪｏｕｒｎａｌｏｆＮｅｕｒｏｌｍｔｈｏｌｏｇｙａｎｄＥｘｐｅｒｉｍｅｎｔａｌＮｅｕｒｏｌｏｇｙ， ２００５，６４（１）：３７－－４５ ２５ＢｒｏｃｋｅｓＪＰ．ＦｉｅｌｄｓＫＬ，ＲａｆｔＭＣ．ＳｔｕｄｉｅｓｏｎｃｕｌｔｕｒｅｄｒａｔＳｏｈｗａｎｎ ｃｅｉｌｓＩ．Ｆ５ｔａｂｌｉｓｈｍｅｎｔｏｆｐｕｒｉｆｉｅｄｐｏｐｕｌｓｔｉｎｎｓｆｒｏｍｃｕｌｔｕｒｅｓｏｆｐｅ－６ｐｈｅｎｄｎｅｔａｍ［Ｊ］．ＪＢｒａｉｎＲｅｓ，１９７９，１６５（１）：１０５－－１１８ ２６ＥｖａｎｓＧＲＰｅｆｉｐ｜幢ｒａｌｎｍ％－ｅⅫｕＩｙ：ａｒｅｖｉｅｗａｎｄａｐｐｒｏａｃｈｔｏｔｉｓｓｕｅｅｎｇｉｎｅｅｒｅｄｃｏｎｓｔｒｕｃｔ５［Ｊ］ＪＡｌｍｔＲｅｃ。２００１，２６３（４）：３９６～４０４ ２７ＥｖａｎｓＧＲＣｈａｌｌｅｎｇｅｓｔｏｎｅｒｖｅｒｅｇｅｎｅｘａｔｉｏｎ［Ｊ］．ＪＳｅｍｉｎｓｕｒｇＯｎ— ｃｏｌ，２０００，１９（３ｊ：３１２－－３１８ ２８ＭｏｅａｂｅｂｉＡ。ＦｄｌｅｒＰ，Ｗｉｈ，ｇＭ，ｅｔｄＥｆｆｅｃｔｏｆａｌｌｎｇｅｎｅｉｅ Ｓｃｈｗａｒｍｃａｌｌｔｒｏｎｓｐｌａｎｔａｔｉｏｎｏｎｐｅｒｉｐｈｅｒａｌｎ口ｗｒｅｇｅａ＿ｌｅｒａｔｉｃｓａ［Ｊ］．Ｊ ＥｘｐＮｅｔｌｒｏｌ，２００２．１７３（２）：２１３－－２２３ ２９Ｒ砾Ｊ，ＫａｐｓＣ，ＢｕｒｍｅｓｔｅｒＧＲ，ｅｔａ１．Ｓｔｅ口ｎｃｅｉｌｓｆｏｒｒｅｇｅｎｅｒａｔｉｖｅｍｅｄｉｃｉｎｅ：ａｄｖａｎｃｅｓｉｎｔｈｅｅｎｇｉｎｅｅｒｉｎｇｏｆｔｉｓｓｕｅｓａｎｄｏｒｇａｎｓ［Ｊ］ＪＮａｍｒｖｄ疆ｅｎｓｃｈａｆｔｅｎ．２００２。８９（８）：３３８～３５１ 组织工程学修复骨缺损的研究进展 广西医科大学第一附属医院创伤骨科手外科（南宁５３００２１）牛通综述赵劲民审校 骨缺损在临床上比较常见，其治疗特别是大段骨缺损的治疗是一个非常棘手的问题。近２０多年来组织工程的发展为骨缺损的修复治疗开辟了一条崭新的道路。在修复骨缺损的过程中涉及到种子细胞、支架材料、细胞因子、组织工程化骨的构建和临床应用等一系列过程。本文就这些方面作一综述。 １种子细胞 种子细胞是组织工程学研究中最基本的问题。组织工程学种子细胞的来源是多渠道的，目前其来源主要有皮质骨、松质骨、骨膜、骨髓、骨外组织以及胚胎干细胞。皮质骨、松质骨、骨膜来源的成骨细胞能表达成骨细胞表型【“，且骨膜中含有较多的骨原细胞，而骨原细胞具有分化潜能可以增值分化为成骨细胞。因此成骨细胞是骨组织工程学研究较多的种子细胞之一。但是上述三者来源的成骨细胞存在较多的缺陷，如取材困难、来源有限、扩增能力有限及免疫排斥等，因而不能稿足骨组织工程的要求。胚胎千细胞具有分化为三个胚层的能力，体外培养后可分化为肠上皮细胞（内胚层）、软骨、骨、 平滑肌、横纹肌（中胚层）及神经细胞（外胚层）等，并且可以大量扩增和定向诱导为具体干细胞，应用这种千细胞可以进行多种移植。Ｂｕｔｔｅｒｙ等¨Ｊ证实用含地塞米松、肛甘油磷酸、维生素Ｃ等培养液或与成骨细胞共同培养均可诱导ＥＳＣｓ向成骨细胞转化，因此胚胎干细胞可以作为骨组织工程学的种子细胞，但是存在免疫排斥较强的缺陷。于是寻找一种取材方便，对机体损伤小；体外培养中具有较强的增值和向成骨方向定向分化的能力；植人体内后能耐受机体免疫排斥，继续保持良好的生物学活性；安全性好的种子细胞变得非常必要。骨髓基质干细胞可以从骨髓中抽取并可以多次抽取，因此它的来源不受限制，取材方便，对供体损伤小，易于分离培养，并且具有体外增殖能力强，大量传代培养后仍具有成骨能力，成为目前应用最广泛的种子细胞。 ２支架材料 支架材料也是组织工程中的重要组成部分，它为种子细胞提供了黏附、增殖、分化的空间结构和生长模板，并且可以引导组织再生，控制组织或器官的性状。支架材料可以有不 万方数据
用于人工骨的材料 目前用于骨修复的生物材料分为以下几种:医用生物陶瓷、医用高分子材料、医用复合材料、纳米人工骨 一．医用生物陶瓷材料 生物活性陶瓷, 主要指磷灰石(AP) ,包括羟基磷灰石(HAP)和磷酸三钙( TCP)等。目前应用最多的是HAP。人骨无机质的主要成分是HAP,它赋予骨抗压强度, 是骨组织的主要承力者,人工合成的HAP是十分重要的骨修复材料,这是由于它的 组成性质与生物硬组织的HAP极为相似,并具有良好的生物相容性,可与自然骨形 成强的骨键合,一旦细胞附着、伸展,即可产生骨基质胶原,以后进一步矿化,形成骨组织。 α2磷酸三钙(α2TCP)骨水泥具有水合硬化特性,可作为一种任意塑型的新型人 工骨用于骨缺损填充。它在动物体内形成蜂窝状结构,动物组织可逐渐长入此蜂 窝状结构中,形成牢固的骨性键合[ 3 ]。β2TCP[ 4 ]属可吸收生物陶瓷,在体内 要被逐渐降解和吸收,但其强度较低,主要用于骨修复或矫正小的骨缺损或骨缺陷, 如骨缺损腔填充。尽管β2TCP植入体内可被降解和吸收,新骨将逐渐替换植 入体,但由于其降解和吸收速度与骨形成速度难达到一致,所以不宜作为人体承 力部件。目前磷酸钙陶瓷要用于作小的承力部件、涂层、低负载的植入体。二．医用生物高分子材料 高分子聚合物已被广泛用作骨修复材料,可降解聚乳酸( PLA)用于口腔外科,聚甲基丙烯酸甲酯( PMMA)骨水泥用于骨填充,聚乙醇酸( PGA)作为可吸收螺 钉用于骨固定。 生物降解材料制作的接骨材料,其弹性模量较金属更接近骨组织的弹性模量,有利于骨折愈合,且随着骨折的愈合,材料逐渐在体内降解,不需二次手术取出。PLA[ 5 ]是一类有应用价值的生物材料,它的降解速度取决于它的分子量、分子 取向、结晶度、物理及化学结构,但其降解的机制主要是因为酯键的水解。目前PLA主要用于骨外科部件,例如骨针、骨板。Minori et al[ 7 ]用不同分子量的PLA 和聚乙二醇( PEG)制成PLA2PEG 共聚物作为骨形成蛋白(BMP ) 的载体, 其 中PLA 6 5002PEG3 000共聚物具有一定的弹性,是较好的BMP载体。 三．医用复合材料 复合人工骨[ 13 ]的研究近年来取得了很大进展,其基本原理是将具有骨传 导能力的材料与具有骨诱导能力的物质如骨生长因子、骨髓组织等复合制备成复合人工骨,使它们既具有骨传导作用,又具有骨诱导作用。 3. 1 磷酸钙复合人工骨主要包括TCP及HAP与胶原、骨生长因子等复合人工骨。原位自体骨与磷酸钙人工骨混合植骨应用在脊柱侧凸畸形矫正术中, 是一种实用、简易、可靠的植骨方法。 3. 2 聚合物复合人工骨生物降解聚合物是近年生物材料研究领域中的一个 热点,通过技术工可合成各种结构形态,一定的生物降解特性的各种聚合物。但 它们无骨诱导活性,需与其它骨诱导因子复合应用才能取得良好效果。 3. 3 红骨髓复合人工骨骨髓由造血系统和基质系统两部分组成。健康红骨 髓的基质细胞中含有定向性骨祖细胞(DOPC)和可诱导性骨祖细胞( IOPC) 。DOPC 具有定向分化为骨组织的能力,IOPC在诱导因子(如BMP)作用下才能分化成骨。
生物材料——骨组织工程讨论组织工程（Tissue Engineering）是近年来正在兴起的一门新兴学科，组织工程一词最早是由美国国家科学基金会1987年正式提出和确定的。它是应用生命科学和工程学的原理与技术，在正确认识哺乳动物的正常及病理两种状态下结构与功能关系的基础上。研究、开发用于修复、维护、促进人体各种组织或器官损伤后的功能和形态生物替代物的科学。 组织工程的核心就是建立细胞与生物材料的三维空间复合体，即具有生命力的活体组织，用以对病损组织进行形态、结构和功能的重建并达到永久性替代。共基本原理和方法是将体外培养扩增的正常组织细胞，吸附于一种生物相容性良好并可被机体吸收的生物材料上形成复合物，将细胞-生物材料复合物植入机体组织、器官的病损病分，细胞在生物材料逐渐被机体降解吸收的过程中形成新的在形态和功能方面与相应器官、组织相一致的组织，而达到修复创伤和重建功能的目的。 骨组织构建 构建组织工程骨的方式有几种：①支架材料与成骨细胞；②支架材料与生长因子；③支架材料与成骨细胞加生长因子。 生长因子通过调节细胞增殖、分化过程并改变细胞产物的合成而作用于成骨过程，因此，在骨组织工程中有广泛的应用前景。常用的生长因子有：成纤维细胞生长因子（FGF）、转化生长因子（TGF-ρ）、胰岛素样生长因子（IGF）、血小板衍化生长因子（PDGF）、
骨形态发生蛋白（BMP）等。它们不仅可单独作用，相互之间也存在着密切的关系，可复合使用。目前国外重点研究的项目之一，就是计算机辅助设计并复合生长因子的组织工程生物仿真下颌骨支架。有人采用rhBMP-胶原和珊瑚羟基磷灰石（CHA）复骨诱导性的骨移植、修复大鼠颅骨缺损，证实了复合人工骨具有良好的骨诱导性和骨传导性，可早期与宿主骨结合，并促进宿主骨长大及新骨形成。用rhBMP-胶原和珊瑚复合人工骨修复兔下颌骨缺损，结果显示： 2个月时，复合人工骨修复缺捐赠的交果优于单纯珊瑚3个月时，与自体骨移植的修复交果无明显差异。 目前，用组织工程骨修复骨缺损的研究，已从取材、体外培养、细胞到支架材料复合体形成等都得到了成功。有人用自体骨髓、珊瑚和rhBMP-2复合物修复兔下颌骨缺损，结果表明：术后3个月，单独珊瑚组及空白对照组缺损未完全修复；珊瑚-骨髓组和珊瑚-rhBMP-2组及单独骨髓组已基本修复了缺损；而骨髓、珊瑚和rhBMP-2复合物组在2个月时缺损即可得到修复。我们用骨基质成骨细胞与松质骨基质复合物自体移植修理工复颅骨缺损的动物实验，也取得了满意的治疗效果。 带血管蒂的骨组织工程是将骨细胞种植于预制带管蒂的生物支架材料上，将它作为一种细胞传送装置。我们将一定形状的thBMP-2、胶原、珊瑚复合物植入狗髂骨区预制骨组织瓣，3个月时，复合物已转变成血管化骨组织。
人工工骨作为用于修复或替换人体硬组织的生物材料，必须具备独特的性能。人工骨材材料与一般工业材料的最大区别在于它们的使用环境不同：人工骨材料是在生物环境内工作，就是说，它要工作在温度为37℃左右、气压为latm*、pH值为7左右的苛刻条件下。所以，人工骨材料不但要具备适度的力学性能，即强度、延伸率、刚度和韧性，而且还要具备生物亲和性、可灭菌性、非毒性、机能性以及耐久性。同时，人工骨材料还必须具备独特微妙的结构，因为天然的骨头是一个多孔而又倾斜的结构体系。 本章将介绍目前人工骨材料的研究现状，特别是近年国际上在人工骨材料研究方面所取得的成果；同时，还将报告作者本人在人工骨材料研究领域所取得的成果。作者研制的多孔钛泡沫具有良好的生物亲和性，无毒，其机械性能与天然骨的机械性能相近。钛泡沫的结构与天然骨的结构一致，其孔空间允许新生骨芽细胞的生长侵人以及体液的传输，所以它不但能与自然骨形成生物性骨键合，与人体骨骼合而为一，而且能诱导新生骨生成，是一种具有良好临床应用前景的骨移植材料。 2.1人工骨材料的种类和特点 生物材料指任何用于治疗的、包括天然的和合成的、与人的细胞直接相接触的材料。人工骨就是用于修复或替换人体硬组织的生物材料。随着人口的急速高龄化，中青年创伤的增加以及天生缺陷和疾病的存在，社会对人工骨材料和医学制品的需求急速增长。老年人最常见的骨质疏松、疾病（如恶性肿瘤切除)、交通事故和火器创伤等都可能造成大型骨缺损。用来修复骨缺损的骨替代材料可以用自体骨移植、人工骨、诱导成骨材料和异体骨移植等，其中以自体骨移植效果最好。但自体骨来源有限，而且可能在供区造成继发性损失或并发症。而现有的人工骨、诱导成骨材料和异体骨移植等均达不到自体骨的效果，为此，进一步寻找尽可能达到或接近自体骨移植效果的理想人工骨材料是对基础研究和临床医学的挑战。 2.1.1 陶瓷材料 人体骨骼主要由胶原质（collagen)和羟基磷灰石(HA)组成，羟基磷灰石的分子式为Ca10（PO4)6(OH)2，其中钙的存在赋予骨骼以强度。骨的结构是一个精致复杂的多孔结构。骨的表层是皮质骨，其孔隙率比较低，约为5％～10％；表层以下是海绵骨，其孔隙率比较高，达50％一90%.骨结构如图2-1所示。骨的力学性能是依个人、年龄和骨的部位而改变的，表2-1所示总结了各种骨的机械性能及强度。 作为人工骨材料，其首要性能当然是在生物体内的耐腐蚀性和强度，但生物亲和性也是不可缺少的重要性能。近年的研究更是注重于人工骨材料的表面处理。 根据其与生物组织的反应，生物陶瓷可以作如表2-2所示的分类：①生物体内惰性型（和自家骨直接接触，也有可能在两者之间介入线纤维皮膜）；圆生物体内活性型（自家骨与人工骨发生化学反应而结合）；圆生物体内分解型（移植后，人工骨在生物体内分解并被新生骨所替代）：作为人工骨材料，力学强度固然重要，然而，骨诱导能力、骨传导能力的具备，进而实现骨骼的修复和新生骨的形成更具魅力p 目前，很多研究都朝着这个方向即开发理想的人工骨材料而努力着。 2.1.2 高分子材料 近年来高分子材料作为人工骨材料也越来越受到重视。高分子材料包括天然的和人工合成的两大类,它们最突出的特点是柔软性、易加工性以及质量轻等。正是因为这些特点，高分子材料被广泛应用于生物领域，如血液的包装、输液系统、导（尿）管、血液回路、血液透析器等一次性使用器具，以及人工肾脏、人工肺、血浆交换膜等人工脏器[ 高分子材料如聚甲基丙烯酸甲酯(PMMA)即骨水泥和用于人工关节的高分子聚乙烯(polyethylene)，这类材料的生物相容性较差，与骨组织之间有纤维组织间隔。 还有一类可生物降解的高分子材料以聚丙交酯（polylactide)和聚乙醇酸(polyglycolide)为代表。目前主要用于可降解内固定材料方面，作为植骨替代材料，多以复合材料的形式出现。
骨组织修复材料的仿生合成 侯京朋 长期以来, 缺损骨骼的再生修复一直是骨研究领域的重要内容。近20年来, 骨的仿生制备已成为缺损骨骼修复研究的重要内容。几乎所有优异的生物矿化材料都采取有机分子调控无机相生长的策略, 因此, 从生物分子调控水平上去理解骨的形成和矿化过程, 并在此基础上研究骨生物材料的合成是突破这一领域的 关键。 1 分子仿生的原理 受天然生物体结构和功能的启发, 采用仿生的思想进行生物材料的合成设计已有悠久历史。传统的仿生学设计, 常采用材料合成的方法去模拟生物体系。但是, 天然矿化组织都是由生物大分子(脂类、蛋白、多聚糖)和无机矿物组成的复合材料, 从宏观到微观、从分子到纳米都是自组装的有序等级结构。这种结构主要是利用有机大分子(蛋白质、多糖、脂类等)自组装, 无机晶体核化、定向、生长和空间形态等方面的调控作用使其在纳米水平上表现出非凡的有序性, 这些都是传统的材料合成方法所无法实现的。随着分子生物学、分子物理、化学和纳米技术的发展, 依据生物矿化过程的“有机基质调控”理论, 生物大分子的自组装和纳米合成技术的联合应用, 使仿生学进入了分子水平, 在此基础上形成一门新的分支学科———仿生材料化学。 2 骨组织修复材料仿生合成的现状 2.1 自组装表面活性剂微囊仿生合成无机骨修复材料 通过表面活性剂形成脂质小泡, 原位合成具有复杂微孔结构和精确表面形态的仿生无机材料。Walsh等首次使用微乳方法合成了高度有序的无机仿生骨材料。刘景洲以天然来源的卵磷脂为双亲分子, 正十四烷油相和水相形成的微乳胶为磷酸钙矿化的“模板”, 调控、诱导矿化。获得由卵磷脂与羟基磷灰石(HA)共同构建的具有纳米结构的立体网状、空心棒状、空心球状产物, 制备了具有纳米微观结构的生物活性替代材料。这些方法主要应用于合成无机生物材料, 而且必须去除表面活性剂。 2.2 钛材表面的仿生涂层
用于人工骨的材料 Revised by Jack on December 14,2020
用于人工骨的材料 目前用于骨修复的生物材料分为以下几种:医用生物陶瓷、医用高分子材料、医用复合材料、纳米人工骨 一．医用生物陶瓷材料 生物活性陶瓷, 主要指磷灰石(AP) ,包括羟基磷灰石(HAP)和磷酸三钙( TCP)等。目前应用最多的是HAP。人骨无机质的主要成分是HAP,它赋予骨抗压强度,是骨组织的主要承力者,人工合成的HAP是十分重要的骨修复材料,这是由于它的组成性质与生物硬组织的HAP极为相似,并具有良好的生物相容性,可与自然骨形成强的骨键合,一旦细胞附着、伸展,即可产生骨基质胶原,以后进一步矿化,形成骨组织。 α2磷酸三钙(α2TCP)骨水泥具有水合硬化特性,可作为一种任意塑型的新型人工骨用于骨缺损填充。它在动物体内形成蜂窝状结构,动物组织可逐渐长入此蜂窝状结构中,形成牢固的骨性键合[ 3 ]。β2TCP[ 4 ]属可吸收生物陶瓷,在体内要被逐渐降解和吸收,但其强度较低,主要用于骨修复或矫正小的骨缺损或骨缺陷, 如骨缺损腔填充。尽管β 2TCP植入体内可被降解和吸收,新骨将逐渐替换植入体,但由于其降解和吸收速度与骨形成速度难达到一致,所以不宜作为人体承力部件。目前磷酸钙陶瓷要用于作小的承力部件、涂层、低负载的植入体。 二．医用生物高分子材料 高分子聚合物已被广泛用作骨修复材料,可降解聚乳酸( PLA)用于口腔外 科,聚甲基丙烯酸甲酯( PMMA)骨水泥用于骨填充,聚乙醇酸( PGA)作为可吸收螺钉用于骨固定。 生物降解材料制作的接骨材料,其弹性模量较金属更接近骨组织的弹性模量,有利于骨折愈合,且随着骨折的愈合,材料逐渐在体内降解,不需二次手术取出。PLA[ 5 ]是一类有应用价值的生物材料,它的降解速度取决于它的分子量、分子取向、结晶度、物理及化学结构,但其降解的机制主要是因为酯键的水解。目前PLA主要用于骨外科部件,例如骨针、骨板。Minori et al[ 7 ]用不同分子量的PLA 和聚乙二醇( PEG)制成PLA2PEG 共聚物作为骨形成蛋白(BMP ) 的载体, 其中PLA 6 5002PEG3 000共聚物具有一定的弹性,是较好的BMP载体。 三．医用复合材料 复合人工骨[ 13 ]的研究近年来取得了很大进展,其基本原理是将具有骨传 导能力的材料与具有骨诱导能力的物质如骨生长因子、骨髓组织等复合制备成复合人工骨,使它们既具有骨传导作用,又具有骨诱导作用。 3. 1 磷酸钙复合人工骨主要包括TCP及HAP与胶原、骨生长因子等复合人工骨。原位自体骨与磷酸钙人工骨混合植骨应用在脊柱侧凸畸形矫正术中, 是一种实用、简易、可靠的植骨方法。 3. 2 聚合物复合人工骨生物降解聚合物是近年生物材料研究领域中的一个 热点,通过技术工可合成各种结构形态,一定的生物降解特性的各种聚合物。但 它们无骨诱导活性,需与其它骨诱导因子复合应用才能取得良好效果。 3. 3 红骨髓复合人工骨骨髓由造血系统和基质系统两部分组成。健康红骨 髓的基质细胞中含有定向性骨祖细胞(DOPC)和可诱导性骨祖细胞( IOPC) 。DOPC具有定向分化为骨组织的能力,IOPC在诱导因子(如BMP)作用下才能分化成骨。Zakrzewska et al[ 17 ]将骨髓细胞与HAP结合,并分别加入成纤维细胞生长因子( bFGF) 和(或) 成骨蛋白21(OP21) ,通过测定胸腺嘧啶掺入到DNA中的量、ALP的活性及新生骨的形成,来了
异种骨和人工骨修复骨肿瘤性骨缺损 方志伟，李舒，樊征夫，白楚杰，刘佳勇，薛瑞峰，张路 北京大学肿瘤医院暨北京市肿瘤防治研究所，骨与软组织肿瘤科，恶性肿瘤发病机制及转化研究教育部重点实验室，北京市 100142 Xenograft and calcium sulphate in treating benign bone tumor Fang Zhi-wei, Li Shu, Fan Zheng-fu, Bai Chu-jie, Liu Jia-yong, Xue Rui-feng, Zhang Lu Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Orthopedic Oncology, Peking University Cancer Hospital & Institute, Beijing 100142, China 摘要 背景：自体植骨是修复骨肿瘤刮除后骨缺损最理想的材料和方法，但存在增加手术创伤，取骨部位的后遗症如感染和疼痛及自体骨的取量有限等缺点。 目的：分析硫酸钙人工骨和异种骨修复良性骨肿瘤刮除后骨缺损的临床疗效。 方法：选择26例良性骨肿瘤患者，其中骨巨细胞瘤8例，内生软骨瘤5例，纤维组织细胞瘤4例，骨纤维异样增殖症3例，非骨化性纤维瘤2例，骨囊肿2例，动脉瘤样骨囊肿和软骨母细胞瘤各1例。12例采用单一硫酸钙骨粒填充肿瘤切除后的骨缺损，6例采用单一异种骨条填充肿瘤切除后的骨缺损，8例采用硫酸钙骨粒+异种骨条填充肿瘤切除后的骨缺损。治疗后1周内、3个月、1年拍X射线片检查，了解植骨愈合情况。 结果与结论：治疗后随访36-72个月，发现硫酸钙骨粒的降解发生较早，一般治疗后1个月就开始出现骨粒降解，3个月大部分已降解完毕并有骨替代发生，1年骨修复塑型良好；异种骨条3个月后降解并有骨替代发生，植骨充填物边缘模糊，6个月后骨缺损及充填物之间边界变模糊，有融合现象，1年骨缺损内密度均匀，骨小梁形成明显，骨修复良好；骨粒+骨条混合植骨者介于单纯硫酸钙骨粒和单纯异种骨条之间，出现骨粒部分先降解先修复、骨条部分后降解后修复，一般术后1年达到骨性愈合。说明硫酸钙人工骨和异种骨在骨肿瘤性骨缺损修复应用中的效果良好，在良性骨肿瘤刮除后植骨可以替代自体骨植骨。 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程 全文链接： 关键词：生物材料；骨生物材料；骨肿瘤；人工骨；硫酸钙；异种骨；植骨； Abstract： BACKGROUND: Autologous bone graft is the best method to repair bone defects after tumor curettage, but its shortcomings are as follows: increased surgical trauma, sequelae at bone graft site such as infection and pain, and a limited amount of autologous bone. OBJECTIVE: To analyze the effectiveness of xenograft and calcium sulphate artificial bone in treating bone defects after benign bone tumor removed. METHODS: Totally 26 cases of benign bone tumor were selected, including 8 cases of giant cell tumor, 5 of enchondroma, 4 of fibrous histiocytoma, 3 of bone fibrous dysplasia, 2 of non-ossifying fibroma, 2 cases of bone cysts, 1 of aneurysmal bone cyst and 1 of aneurysmal bone cyst and 1 case of chondroblastoma. Of the 26 cases, 12 cases underwent calcium sulphate pellets alone to fill bone defects after benign bone tumor removed, 6 cases were subjected to xenograft alone, and 8 cases were treated with calcium sulphate pellets combined with xenograft. The X-rays were taken at 1 week, 3 months, and 1 year after the operation in all patients to assess the bone healing process. RESULTS AND CONCLUSION: All the patients were followed up for 36-72 months. The absorption of calcium sulphate appeared to be absorbed earlier, the earlier absorption appearance could be observed as earlier as 1