Therapeutic bioactive microcarriers:Co-delivery of growth factors and stem cells for bone tissue
engineering
R.A.Perez a ,A.El-Fiqi a ,b ,c ,J.-H.Park b ,T.-H.Kim a ,J.-H.Kim a ,H.-W.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 Research Center,Dankook University,Cheonan 330-714,South Korea c
Glass Research Department,National Research Center,Egypt d
Department of Biomaterials Science,School of Dentistry,Dankook University,Cheonan 330-714,South Korea
a r t i c l e i n f o Article history:
Received 2May 2013
Received in revised form 14September 2013Accepted 30September 2013Available online 9October 2013Keywords:Microcarriers
Bone tissue engineering Growth factor delivery Sustainable release Bioactive glass
a b s t r a c t
Novel microcarriers made of sol–gel-derived bioactive glasses were developed for delivering therapeutic molecules effectively while cultivating stem cells for bone tissue engineering.Silica sols with varying concentration of Ca (0–30mol.%)were formulated into microspheres ranging from 200to 300l m under optimized conditions.A highly mesoporous structure was created,with mesopore sizes of 2.5–6.3nm and specific surface areas of 420–710m 2g À1,which was highly dependent on the Ca concentration.Ther-apeutic molecules could be effectively loaded within the mesoporous microcarriers during microsphere formulation.Cytochrome C (cyt C),used as a model protein for the release study,was released in a highly sustainable manner,with an almost zero-order kinetics over a period of months;the amount released was $2%at 9days,and 15%at 40days.A slight increase in the release rate was observed in the micro-carrier containing Ca,which was related to the dissolution rate and pore size.The presence of Ca accel-erated the formation of hydroxyapatite on the surface of the microcarriers.Cells cultured on the bioactive microcarriers were well adhered and distributed,and proliferated actively,confirming the three-dimensional substrate role of the microcarriers.An in vivo study performed in a rat subcutaneous model demonstrated the satisfactory biocompatibility of the prepared microspheres.As a therapeutic target molecule,basic fibroblast growt
h factor (bFGF)was incorporated into the microcarriers.A slow release pattern similar to that of cyt C was observed for bFGF.Cells adhered and proliferated to significantly higher levels on the bFGF-loaded microcarriers,demonstrating the effective role of bFGF in cell prolifer-ative potential.It is believed that the developed mesoporous bioactive glass microspheres represent a new class of therapeutic cell delivery carrier,potentially useful in the sustainable delivery of therapeutic molecules such as growth factors,as well as in the support of stem cell proliferation and osteogenesis for bone tissue engineering.
Ó2013Acta Materialia Inc.Published by Elsevier Ltd.All rights reserved.
1.Introduction
The release of specific signaling molecules from scaffolding materials to elicit desired cellular reactions is one of the key strat-egies to significantly enhance the regenerative capacity of syn-thetic biomaterials for use in bone tissue engineering.These signaling molecules can either be eluted to the cells that are sup-ported upon the scaffolds for ex vivo cultivation appropriate for bone engineering,or can be supplied to the surrounding bone de-fects when directly implanted in vivo [1].For either case,the sig-naling molecules should ideally be released in a controlled
钱伯manner,and for certain durations,in order to optimize cellular propagation and/or osteogenic differentiation,as well as to aid in vivo vascularization and bone formation.Hence,one of the pri-mary requirements of scaffolds is the sustained and controllable release of therapeutic molecules,while enabling their effective and safe incorporation into the scaffold structure.
Microspherical scaffolds,namely,microcarriers,have gained great interest as three-dimensional (3-D)substrates for the 3-D cultivation and expansion of tissue cells.Frequently allowed to ro-tate in cell suspensions in vitro,so as to aid cellular anchorage,the isotropic 3-D spherical substrates provide homogeneous sites for cellular recognition,distribution and multiplication [2].Subse-quently,cell-loaded microcarriers can be delivered to the defects of concern,with a delivery capacity that is controllable based on the size of the microcarriers.Furthermore,the cell–carrier constructs are possible as injectable tissue engineering devices,
1742-7061/$-see front matter Ó2013Acta Materialia Inc.Published by Elsevier Ltd.All rights reserved./10.1016/j.actbio.2013.09.042
⇑Corresponding author at:Institute of Tissue Regeneration Engineering (ITREN),Dankook University,Cheonan 330-714,South Korea.Tel.:+82415503081;fax:+82415503085.
E-mail address:kimhw@dku.edu (H.-W.Kim).
effectivelyfilling defects[3].In fact,the micro-spherical particles, which may be a few to hundreds of micrometers in diameter,have been extensively researched for the delivery of therapeutic mole-cules,including drugs,hormones and growth factors[4,5].Along with this biomolecular delivery use,the cellular delivery potential has thus been of great interest for the ex vivo culture of stem cells and engineering of tissues,including bone.
Among other things,providing therapeutic roles to the micro-carriers is of great merit in regulating the behavior of stem/ progenitor cells to be supported,ultimately for bone tissue engineering,such as rapid cellular engulfment,increased cell population,stimulation to osteogenic lineage specification and/or achieving highly vasculature tissues[2].When microcarriers are used for the delivery of therapeutic molecules,cells can be made to undergo osteogenesis during ex vivo cultivation prior to implan-tation and,further,to play beneficial roles in the regeneration process of bone tissue in vivo,after implantation.
Therefore,the current aim is to develop therapeutic microcarri-ers effective for bone tissue engineering that allow3-D cultivation of stem cells in vitro,while incorporating and releasing therapeut
黎祥ic molecules ultimately to aid their osteogenesis and in vivo bone for-mation.Here,the present authors propose a‘‘bone-bioactive’’inor-ganic composition composed of silica-based bioactive glass(SBG). The SBG is made through a sol–gel process under room tempera-ture and aqueous conditions[8–12].Different ions could be easily incorporated into the silica sol–gel glass network,and the addition of calcium greatly improved the bioactivity and hydrolytic degra-dation.Natural polymers such as gelatin and collagen have also been added,to improve the mechanical properties and cellular re-sponses[13–15].Importantly,the sol–gel process enables the introduction of therapeutic molecules because of the mild process-ing conditions.Several studies have demonstrated the effective-ness of the sol–gel glass network in capturing drugs and proteins and their release for long periods[16–20].
Here,the SBG composition is used in the preparation of thera-peutic microcarriers,which are effective for bone tissue engineer-ing.The SBG composition has merits over polymer-based compositions,particularly for therapeutic purposes,which can limit the incorporation of biomolecules.Synthetic polymers are generally produced in organic solvents or surfactant-mediated conditions,requiring vigorous washing steps,while natural poly-mers require crosslink steps to stabilize the structure,although they may be processed in aqueous solutions[2].Moreover,the SBG co
mposition based on a sol–gel process has a number of mer-its compared with conventional bioceramics,such as calcium phosphates and melt-derived bioglasses,that are generally prepared at high temperatures[6,7].Owing to the nature of the sol–gel ,hydrolysis and polycondensation,sol–gel pro-cessed BG spherical particles self-harden in a structurally and chemically stable manner,eliminating further crosslinking steps.
Another intriguing and beneficial point of SBG is the sol–gel-derived mesoporous structure,where the mesoporosity and mesopore geometry are tuned to take up a large quantity of and selective therapeutic molecules and,subsequently,to control their release behavior[16–18].Furthermore,the silanol groups present on the surface are hydrophilic and easily inducible for calcium and phosphate ions to produce calcium phosphate crystals,which are recognized as‘‘bone-bioactive’’materials[21],and therefore SBG is considered to be a proper reservoir for therapeutic mole-cules and3-D substratum for cellular reactions,particularly those required for bone regeneration.
In the present study,SBG microcarriers with bone-bioactive and self-hardening properties are prepared.The ability to populate stem cells in vitro,as well as to allow favorable reactions in vivo, is briefly assessed.Model experiments on incorporating therapeu-tic protein molecules within the struct
ure and releasing them sustainably,as well as the accompanying biological effects,are also described.These studies support the further use of novel therapeu-tic microcarriers in bone tissue engineering.
2.Materials and methods
2.1.Preparation of SBF microcarriers
Tetraethyl orthosilicate(TEOS,C8H20O4Si,98%,Sigma–Aldrich) (10ml)was mixed with0.1M HCl(2.4ml),with the addition of deionized water to form an acid catalyzed sol.The molar ratio of total water(including the CaCl2and the HCl water)to TEOS was8.The microspheres were doped with calcium ions by incorpo-rating specific amounts of calcium chloride(CaCl2,Sigma–Aldrich) into the solution.The molar percentage of doped calcium ranged from0to30.Once the sol was obtained,it was cooled down in a water bath at4°C.Afterwards,0.08M ammonium hydroxide (NH4OH,28.0%NH3in water,P99.99%metal basis,Sigma–Aldrich) was added dropwise to the sol with agitation.The pH was adjusted to5–5.5,and5ml of the sol was then added dropwise to100ml of olive oil,and was stirred at95rpm to allow gelation.Gelled micro-spheres were gathered after precipitation at the bottom of the flask,vacuumfiltered,rinsed with water and ethanol,and left over-night to dry.Microcarriers with differe
nt Ca concentrations were designated as‘‘0Ca’’,‘‘10Ca’’,‘‘20Ca’’and‘‘30Ca’’for0%Ca–100%Si, 10%Ca–90%Si,20%Ca–80%Si and30%Ca–70%Si,respectively.
2.2.Characterizations of microcarriers
The size distribution of the microspheres was characterized by means of laser diffraction granulometry(Malvern,APA5001SR). Specific surface area as well as the pore size distribution was quan-tified using a Quadrasorb SI automated surface area and pore size analyzer(Quantachrom Instruments).Samples were degassed un-der vacuum at300°C for12h prior to analysis.The specific surface area was determined according to the Brunauer–Emmett–Teller (BET)method.The pore size distribution was determined from the N2desorption branch of the N2adsorption–desorption iso-therms obtained on the basis of the density functional theory method.Total pore volume was calculated from the amount ad-sorbed at a maximum relative pressure(P/P0).Scanning electron microscopy(SEM)images were taken to observe the morphology and particle size(JEOL JSM-6510).X-ray diffraction(XRD;Philips MRD)was conducted to analyze the phases.X-rays were generated at40mA and40kV,and data were obtained at diffraction angles (2h)from4to45°,with a step size of0.02°and a scanning speed of5°minÀ1.The chemical bond status in the microspheres was analyzed by Fourier transform infrared spectrometry(FTIR;Varian 640-IR).The f potential of the micro
spheres was measured using a Zetasizer Nano ZS laser Doppler electrophoresis instrument (Malvern Instruments).The f potential was measuredfive times at25°C with an appliedfield strength of20V cmÀ1(where each data point is the average of40runs),and the mean±standard deviation(n=5)were calculated.The instrument automatically calculated electrophoretic mobility(U),and f potential according to the Helmholtz–Smoluchowski equation:f=U g/e,where f is the zeta potential,U is the electrophoretic mobility,g is the dispersing medium viscosity,and e is the dielectric constant.
2.3.In vitro apatite forming ability
The in vitro apatite forming ability of the microcarriers was evaluated through the formation of apatite on the surface of the microspheres when immersed in a simulated bodyfluid(SBF) solution.One hundred milligrams of microcarriers with diameters
R.A.Perez et al./Acta Biomaterialia10(2014)520–530521
between200and500l m were incubated in50ml of concentrated SBF(2ÂSBF)to speed up the reaction and shorten the observation time.The2ÂSBF was buffered at pH7.4with50mM Tris and 45mM HCl,and the temperature was kept constant at37°C.After immersion for3and7days in the solution,the
microcarriers were separated from the solution byfiltration,and were then gently rinsed with distilled water to remove any debris.The formation of apatite on the surface of the microspheres was then analyzed by XRD and SEM.
2.4.In vitro loading and release study;using model protein cytochrome C
The capacity of the microcarriers to incorporate biological mol-ecules,as well as their ability to release them in a sustainable man-ner,was assessed using a model protein cytochrome C(cyt C),as it has a molecular size and charge properties similar to those of bio-logical growth factors[22].Cyt C(Sigma–Aldrich)was encapsu-lated within the microcarriers during the preparation of the microcarriers.For this,cyt C wasfirst dissolved in distilled water, and was then mixed with the initial sol at a ratio of 2.14mg cyt C gÀ1TEOS.After the sol–gel reaction,as described in the previous section,the cyt C-loaded microcarriers were gathered and dried for further use.For the release test,100mg of the cyt C-loaded microcarriers were introduced to each well of24-well plates,containing1ml of phosphate buffered saline(PBS).After incubation at37°C,the supernatant solution was sampled at predetermined time points,and was assayed with a UV spectro-photometer at408nm.At each time point,PBS was replenished prior to the next incubation.The release profile of cyt C from the microcarriers was monitored over a period of6weeks.
2.5.Cell behavior on microcarriers
The ability of the microcarriers to aid the initial anchorage and subsequent proliferation of tissue cells during in vitro culturing was investigated using pre-osteoblast M3CT3-E1cells.The cells were maintained in a-minimal essential medium(a-MEM)(Gibco, Invitrogen)supplemented with2%penicillin/streptomycin(Gibco) and10%fetal bovine serum(Gibco)in a humidified atmosphere of 5%CO2in air.The culture medium was exchanged every2days. Upon confluence,the cells were detached with a minimum amount of trypsin–EDTA(Gibco)after inactivation with FBS,and were then sub-cultured or used for further experiments.
Approximately40mg of microcarriers were placed onto a Trans-well membrane,which was contained in each well of24-well plates to form a monolayer covering the membrane surface,and20,000 cells per well were seeded on top of the microcarriers.After1,3 and7days,the cell number was quantified using a cell counting kit(CCK-8,Dojindo Molecular Technologies).CCK-8is based on the WST-8reaction,which produces an orange formazan dye,the production of which is directly related to the number of viable cells. At each time point,the medium was replaced with200l l of serum-free medium,and was followed by the addition of20l l of CCK-8 solution.The reagent was left to react for2h,and the reactant was then read at450nm using a UV–vis spectrophotometer.
Cell morphology was observed by SEM and confocal laser scan-ning microscopy(CLSM;LSM510,Zeiss).Samples for SEM were initially washed in phosphate buffer0.1M,pH7.4,fixed with 2.5%glutaraldehyde(Sigma–Aldrich G400-4)solution in PBS,and were then washed and maintained in0.1M phosphate buffer.Sam-ples were dehydrated with ethanol solutions in serial concentra-tion(50,70,90,95and100%ethanol)and then treated with hexamethyldisilazane(Fluka52620).Dried samples were mounted on a copper block,gold-coated,and then visualized with SEM.For CLSM observation,at each culture period,the cells werefixed with 4%paraformaldehyde solution and stained with Alexa Fluor546 conjugated Phalloidin(Molecular Probes)and Prolong Gold antif-ade reagent with DAPI(Molecular Probes),after which thefluores-cence signals were visualized.
2.6.In vivo subcutaneous implantation
In vivo tissue reactions of the microcarriers were briefly as-sessed using rat subcutaneous tissues.Two-week-old rats were used for the study.Animal surgery was conducted in accordance with the Animal Care and Use Committee of Dankook University, South Korea.Animals were anaesthetized by intramuscular injec-tion of ketamine/xylazine.The skin on the dorsal region of the rat was then shaved and disinfected with povidone iodine and 70%ethanol.Next,a skin incision was
made using a#10blade with a Bard–Parker scalpel,after which four small subcutaneous pockets were made on the back,and laterally from the spine of each rat,by blunt dissection with baby Metzenbaum scissors.Microcarriers were implanted into the prepared area,and the incision was su-tured using monofilament suture(Prolene).During surgery,the mice were kept warm,while under observation until they recov-ered from the anesthesia.After recovery,the mice were housed and provided with standard pellet food and water ad libitum.
The animals were killed at2weeks after surgery.For histologi-cal samples,the tissue constructs were harvested as a single mass from the rat.The specimens were then immediately immersed in 10%buffered neutralized formalin for24h and were dehydrated with a graded ethanol series(70,80,90,95and100%).Next,the samples were bisected and embedded in paraffin,after which the blocks were serially cut into sections$5l m thick,using a micro-tome(LEICATM)and mounted on microscope slides.Slides with tissue sections were deparaffinized and hydrated through xylene and graded ethanol series,which was subsequently stained with hematoxylin and eosin(H&E)and visualized by optical microscopy.
2.7.Loading and delivery study of bFGF
As a therapeutic molecule,basicfibroblast growth factor(bFGF) was introduced to examine the loading and delivery capacity of the prepared microcarriers.Recombinant human bFGF was obtained as previously described[23].Briefly,the cDNAs of the bFGF were amplified from an adult human cDNA library.According to the GenBank sequence(GeneBank accession number NM002006), a pair of primers,50-CGAGATCTCAGCCGGGAGCATCAC-30and 50-TGCAGATCTCGCTCTTAGCAGACATTG-30,were designed and used.The PCR products were cloned into pBAD/His A(Invitrogen, Carlsbad,CA)in-frame,using the NH2-terminal6X His tag.The recombinant bFGF protein containing the poly-His tag was expressed and purified using a Ni affinity column under denaturing conditions,according to the manufacturer’s protocol(Invitrogen).
Microcarriers were prepared as described in the previous sec-tion,by incorporating within the sol at0.3mg bFGF gÀ1of TEOS by weight.In order to measure the release of bFGF from the micro-carriers,100mg of microcarriers were placed in each well of a 24-well plate,which contained1ml of PBS.At each predetermined time point,the supernatant was gathered and was assessed by bFGF enzyme-linked immunosorbent assay(ELISA,Perrotech). The release assay was continued for7weeks,and at each time point the PBS was refreshed.
2.8.Biological effects of bFGF release on mesenchymal stem cells
The biological effects of the released bFGF from the microcarri-ers were examined on the mesenchymal stem cells(MSCs)derived from rat bone marrow.MSCs were harvested from the femora and tibiae of adult rats(180–200g),according to the guidelines
522R.A.Perez et al./Acta Biomaterialia10(2014)520–530
approved by the Animal Ethics Committee of Dankook University. The harvested product was then centrifuged,and the supernatant was collected and suspended within a cultureflask containing a normal culture medium;a-MEM supplemented with10%fetal bo-vine serum(FBS),100U mlÀ1penicillin and100mg mlÀ1strepto-mycin,in a humidified atmosphere of5%CO2in air at37°C. After incubation for1day,the medium was refreshed and cultured until the cells reached near confluence.After subculture and main-tenance under normal culture conditions,cells at two to three pas-sages were used for further tests.
The effects of bFGF release on the MSC proliferation were as-sessed by the two different sets of experiments;one is direct and the other is an indirect assay.For the direct assay,40mg of micro-spheres contained in a Transwell membrane were inserted into each well of24-well plates,and20,000MSCs were directly seeded on top of the microspheres.Cells were cultured for up t
北京市委书记刘奇o7days in the starvation media(1%FBS).Cell viability was quantified with a cell counting kit assay(CCK-8reagent).For the indirect assay,the microcarriers contained in the Transwell membrane were allowed to interact with the MSCs,which were seeded in each well of 24-well plates.In particular,microcarriers were pre-immersed in culture medium for prolonged periods of either14days or21days, and then the culture medium was refreshed,after which the MSCs were populated at6000to interact indirectly with the microcarri-ers.After additional culture for5or7days in starving conditions (1%FBS),the cell numbers were counted by the CCK assay.Cell counting was performed in triplicate for all experiments.
2.9.Statistical analyses
Data are presented as mean±1standard deviation.Statistical analysis was carried out,using one-way analysis of variance,and significance was defined at P<0.05.
3.Results
3.1.Characteristics of microcarriers
聂鲁达Fig.1a presents the typical SEM morphology of the microcarri-ers.Spherical microparticles were succ
essfully generated for all compositions(shown as representative images for0Ca and30Ca). The size distribution of microcarriers(Fig.1b)shows the genera-tion of microparticles primarily with diameters of hundreds of micrometers,and the incorporation of calcium slightly increased the mean diameter of microcarriers from$200l m for0Ca to $300l m for30Ca.The XRD patterns illustrate all the compositions being completely amorphous with silica glassy phase at2h$22°, which also show a slightly decreased peak intensity with the incor-poration of calcium(Fig.1c)resulting from the reduced silica glassy structure due to calcium disrupting the silica network.The FTIR spectrum shows typical chemical bands related to silica-based glass for all the compositions(Fig.1d):Si–O–Si bending at 478cmÀ1,Si–O–Si asymmetric stretching at819cmÀ1,Si–O–Si symmetric stretching at1103cmÀ1and1230cmÀ1,and Si–OH stretching at971cmÀ1.A broad band at3484cmÀ1was assigned to O–H stretching vibration,and the peaks at2888and 2962cmÀ1correspond to asymmetric and symmetric C–H stretch-ing.No methoxy groups were noticed,indicative of a complete reaction of the initial reagents[14,15].
Because of the sol–gel reactions,the SBG microcarriers possess a high level of mesoporosity.The mesoporous characteristics were evaluated in terms of N2adsorption/desorption isotherms(Fig.2a) and pore size distributions(Fig.2b).The adsorption/desorption iso-therms exhibited hysteresis loops,a
nd the microcarriers contain-ing Ca appeared to show type IV isotherm,which is typically observed in mesoporous materials according to the IUPAC (Internationa Union of Pure and Applied Chemistry)classification. For0Ca,the hysteresis loop is akin to a type I isotherm.The pore size distribution showed a clear dependence on the composition. Increasing the calcium content increased the pore size.The average pore size of0Ca was2.2nm,which was increased to6.3nm for 30Ca.The different hysteresis loop behaviors observed also reflect this difference in the pore size.Table1summarizes the mesopore characteristics of the microcarriers,including pore size,pore vol-ume and surface area.The results show that the incorporation of calcium increased the pore size and specific pore volume (0.34cm3gÀ1for0Ca vs.0.53cm3gÀ1for30Ca),while it corre-spondingly decreased the specific surface area(713m2gÀ1for 0Ca vs.417m2gÀ1for30Ca).The calcium-containing microcarriers possess bigger pores and a higher pore volume,while having a lower surface area,as depicted in Fig.2c.The surface charge property of the microcarriers was also examined by measurement of the f potential.As presented in Table1,the f potential of0Ca was highly negative(À39mV),which shifted to less negative values with calcium incorporation(À9mV for30Ca),which was due to the negatively charged silica network becoming neutralized by the incorporation of Ca2+into the structure.
The in vitro apatite-forming ability of the SBG microcarriers was assessed during immersion in SBF for3days,as presented in Fig.3. In0Ca,mineral deposits were initiated in some areas,as observed by SEM(arrowed,Fig.3a),which,however,did not give clear crys-talline phase development in XRD pattern(Fig.3c).In contrast,in 30Ca,mineral deposits were clearly noticed,with complete cover-age of the surface of microcarriers(Fig.3b).The XRD pattern con-firmed the development of a peak at2h=32°,which corresponds to the apatite phase,mainly in a poorly crystallized form(Fig.3d).
3.2.Protein delivery and biological assessments
硅酸铝纤维毡
The incorporation of biological molecules within the microcar-riers and their release patterns were investigated.First,cyt C was used as a model protein,which was incorporated within the SBG microcarriers during the preparation stage,and the release pattern was examined.Fig.4shows the release profiles of the cyt C incor-porated within0Ca and30Ca microcarriers.Cyt C release was highly sustainable over the test period of6weeks.The release was as little as2%during7days,with almost zero-order kinetics observed,and became$11%for0Ca and14%for30Ca after 42days.Results demonstrate a great potential of the SBG micro-carriers for the long-term sustainable delivery of protein molecules.
The cell scaffolding capacity of the BSG microcarriers was examined by culturing osteoblasts upon the carriers.Fig.5a shows the cell proliferation on the microcarriers(0Ca and30Ca)for up to 7days,as quantified by CCK-8reagent.Cells proliferated actively for both microcarriers.Initially(day1),similar cell numbers ad-hered to the microcarrier surface.After3days,cells on0Ca prolif-erated slightly more,but after7days,cell proliferation was significantly higher on30Ca.SEM(Fig.5b)and CLSM images (Fig.5c)of cells grown on the microcarriers at each culture time also show the highly proliferative cell morphologies,reflecting the cell proliferation rate data.Cells were moreflattened and had better cytoskeletal extensions with prolonged culture time,and upon the Ca-containing microcarriers.The results illustrate that the microcarriers provide effective3-D scaffolding conditions for cells to anchor to,spread and multiply,which is a prerequisite for using the carriers in bone tissue engineering.Along with the in vitro cellular behavior,the in vivo tissue compatibility of the SBG microcarriers was briefly assessed in rat subcutaneous tissue.After2weeks of implantation,H&E-stained images were histologically examined(Fig.5d).Microcarriers showed a high
R.A.Perez et al./Acta Biomaterialia10(2014)520–530523
population of fibroblastic cells gathering around tion with the surrounding tissue,while exhibiting inflammatory cells,demonstrating good tissue After confirming the protein delivery capacity,and tissu
e compatibility of the developed SBG present authors next applied a therapeutic protein Fig.6shows the release profile of bFGF from the ers.bFGF release was highly sustainable over 5weeks,exhibiting near zero-order kinetics.The released was $5%at 7days and $20%at 35days,dence of a plateau effect,which is indicative thereafter.
The biological effects of bFGF released from ers were further addressed,based on the stimulation erative potential.MSCs were directly seeded onto microcarriers (30Ca)and were observed by CLSM ture periods (Fig.7a).Cells cultured on the bFGF-loaded riers exhibited more cytoskeletal extensions,microcarrier surface more profoundly.Based on the fraction of cells either spread or not was For bFGF-free microcarriers,the fraction of $30%at 2days,which became $55%at 7days.of cells were shown to spread at 2days,and came spread at 7days.As well as the cell spreading cell proliferation rate was also stimulated by the microcarriers,with a significant difference and 7(Fig.7c).
The effects on the MSC responses of bFGF longed period were again examined by an indirect microcarriers contained in Transwell membranes from the MSCs seeded on the culture well,where molecules were free to communicate with MSCs.longed pre-immersion of the microcarriers (either the MSCs were cultured for an additional 5morphology and (b)size distribution of the SBG microcarriers with different compositions.(c)XRD pattern and (d)FT-IR spectrum of particles with hun
dreds of micrometers in size was generated for all compositions.Incorporation of calcium increased the an amorphous phase with chemical bands related with silica-based glasses.
(a)N 2adsorption/desorption plot and (b)pore size distribution of the SBG microcarriers with different composition.(c)Schematic representation showing the presence of mesopores within the microcarriers,comparing the different pore size,volume and surface area between 0Ca and 30Ca.While the 30Ca possesses pores and a higher pore volume,0Ca has a higher surface area.
indirect interactions with the microcarriers.Cell proliferation was significantly stimulated by the bFGF-loaded microcarriers for all culture conditions designed over the long pre-immersion periods (Fig.8a),due to the effects of a sustained release of bFGF.Fluores-cence images showed more extended cytoskeletal processes of cells when affected by the bFGF released from the microcarriers (Fig.8b).
4.Discussion
马克思唯物史观
Tissue engineering scaffolds,primarily guiding3-D physical substrate conditions to stem cells,can also provide biochemical signaling cues to regulate cellular behavior
achieve a tissue mimic structure.Of the various
folds currently under development or in use,
microspherical particles to provide3-D scaffolding
stem cells to anchor to and propagate on,
ogenic processes in concert with the delivery
cules.This combined function of delivering
therapeutics is considered ideal to achieve
lents by ex vivo culturing,as well as to help
tion after implantation.
In particular,targeting hard tissues,SBG
bioactive inorganic composition.Furthermore,
duced through a sol–gel reaction.This process,
aqueous room temperature conditions,enables
ration of therapeutic molecules,such as growth
side,the composition self-hardens,resulting
physicochemical stability[24].The sol–gel
densation reactions were well implemented
water-in-oil emulsification.The microspherical
possessed high physicochemical integrity,allowing stable incorpo-ration of water-soluble bioactive molecules within the structure, and ease of separation of the microcarriers via a simple washing step.In particular,the highly mesoporous structure generated dur-ing the sol–gel process is beneficial for loading biological mole-cules and for providing channels for sustainable and controllable release[24].
To this sol–gel-based microcarrier structure were added various amounts of calcium(up to30wt.%)as the incorporated calcium has positive effects on bone bioactivity,such as bone cell prolifer-ation and
osteogenic differentiation as well as apatite mineraliza-tion.A brief examination of the SBF-immersion test demonstrated a significant improvement in the rate of apatite min-eralization on the surface(Fig.3).While the sol–gel-produced SBG
the apatite forming ability of the SBG microcarriers(0Ca and30Ca)during immersion in SBF for7days at37°C:(a,b)
immersion.While the mineral deposition was limited on the0Ca surface,substantial mineral induction was noticed on
observed.The mineral phase showed a peak at2h–32°,typical of apatite in poorly crystallized state.
Release profile of the model protein cyt C,which was incorporated during
sol–gel processing of the microcarriers.Both microcarriers presented a highly
sustained release of cyt C for the test period(6weeks),with a near zero-order
The release profile was affected by the different mesopore characteristics.
R.A.Perez et al./Acta Biomaterialia10(2014)520–530525
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