ODONTOGENIC CELL CULTURE IN PEGDA HYDROGEL...

ABSTRACTIn order to obtain a tooth-like structure, embryonic oral ectodermcells (EOE) and bone marrow-derived stem cells (BMSC) werestratified within a synthetic hydrogel matrix (PEGDA) andimplanted in the ileal mesentery of adult male Lewis rats. Whole-mount in situ hybridization was used to evaluate the expressionof Pitx2, Shh and Wnt10a signals indicative of tooth initiation. Inrats, expression of the three markers was present in the oral ecto-derm starting at embryonic stage E12.5, which was thereforeselected for cell harvesting. Embryos were obtained by controlledservice of young female Lewis rats in which estrus was detectedby impedance reading. At E12.5, pregnant rats were humanelyeuthanized and embryos were collected. The mandibular segmentof the first branchial arch was dissected and the mesenchyme sep-arated from the ectoderm by enzymatic digestion with pancreatintrypsin solution. BMSCs were collected by flushing the marrowof tibiae and femurs of adult Lewis rats with α-MEM and cul-tured in α-MEM in 25 cm2 flasks. Second passage BMSC’s wererecombined with competent oral ectoderm (E12.5-E13) stratify-

ing them within a 3D PEGDA scaffold polymerized by exposureto UV (365nm) inside a pyramidal polypropylene mold. Con-structs were incubated from 24 to 48 hrs in α-MEM and thenimplanted for four to six weeks in the mesentery of adult male (3-6 month old) Lewis rats. 76 constructs were implanted (37 exper-imental, 27 negative controls and 12 positive controls). Uponmaturation, constructs were harvested, fixed in buffered forma-lin, processed and stained with hematoxylin eosin (HE). Histo-logical evaluation of the experimental and negative constructsshowed that BMSCs underwent an apoptotic process due to lackof matrix interactions, known as anoikis, and were thus incapableof interacting with the competent ectoderm. In contrast, embry-onic oral ectoderm was able to proliferate during the mesentericimplantation. In conclusion, PEGDA scaffolds are incompatiblewith BMSCs, therefore it is essential to continue the search for anideal scaffold that allows proper tissue interactions.

Keywords: Polyethyleneglycol diacrylate - 3D scaffolds -embryonic structures - stem cells - mesentery.

243

Vol. 25 Nº 3 / 2012 / 243-254 ISSN 0326-4815 Acta Odontol. Latinoam. 2012

ODONTOGENIC CELL CULTURE IN PEGDA HYDROGEL SCAFFOLDS FOR USE IN TOOTH REGENERATION PROTOCOLS

Lorenza Jaramillo1, Ignacio Briceño2, Camilo Durán1

1 Dental Research Center, School of Dentistry, Pontificia Universidad Javeriana, Colombia2 Genetics Institute, School of Medicine, Pontificia Universidad Javeriana, Colombia.

CULTIVO DE CÉLULAS ODONTOGÉNICAS EN SOPORTES DE HIDROGEL PEGDA PARA USO EN REGENERACIÓN DENTAL

RESUMENPara lograr la formación de una estructura similar a un dien-te se estratificaron células de ectodermo oral embrionario(EOE) con células troncales de medula ósea (BMSC) dentrode una matriz de diacrilato de polietilenglicol (PEGDA) quese implantó en el mesenterio ileal de machos adultos de ratasLewis. Mediante hibridización in situ de bloque completo seevaluó la expresión de tres genes iniciadores putativos de laformación dental (Pitx2, Shh y Wnt10a), estableciendo que enratas las señales iniciadoras de dentogénesis aparecen entreE12.5 y E13. El tejido ectodérmico embrionario se obtuvohaciendo cruces controlados de hembras a las que se les detec-tó el estro mediante impedanciometría. En E12.5 las hembrasse sacrificaron y se extrajeron los embriones. Se disecó la por-ción mandibular del primer arco branquial y el ectodermo seseparó del mesénquima mediante disociación enzimática conuna solución de pancreatina tripsina. Las BMSC se extraje-ron de los huesos largos de las extremidades inferiores deratas mediante lavado con α-MEM y se cultivaron en cajas de25cm2 hasta un segundo pasaje. Las BMSC fueron recombi-nadas con ectodermo embrionario competente (E12.5 - E13)estratificándolas en un soporte tridimensional de PEGDA,polimerizado con luz ultravioleta (365nm) dentro de un molde

piramidal de polipropileno (PP). Los constructos se cultiva-ron entre 48 y 72 horas en α-MEM y posteriormente fueronimplantados en el mesenterio ileal de machos adultos (3 a 6meses de edad) por un período de cuatro a seis semanas. Seimplantaron 76 constructos (37 experimentales, 27 controlesnegativos y 12 controles positivos). En la fecha determinadalos animales se sacrificaron mediante asfixia con una mezclade CO2 y aire recuperando los constructos que se fijaron enformalina tamponada para luego procesarlos y teñirlos conhematoxilina eosina (HE). La evaluación histológica de losconstructos experimentales, positivos y negativos mostró quelas BMSC incluidas en el hidrogel sufrieron un proceso deapoptosis conocido como anoikis que impidió su interaccióncon las células ectodérmicas. En contraste el EOE proliferódurante el período de implantación. A futuro se debe buscarla matriz portadora ideal que permita el confinamiento de losdos grupos celulares y que brinde el soporte estructural nece-sario para la proliferación de las BMSC facilitando su inter-acción con las células inductoras de origen ectodérmico.

Palabras clave: Diacrilato de Polietilenglicol (PEGDA) -soporte tridimensional - estructuras embrionarias - célulasmadre - mesenterio.

ACTA-3-2012-FINAL:3-2011 16/04/2013 11:56 a.m. Página 243

INTRODUCTION

The need to restore or replace deteriorated or lostorgans as a result of alterations in their formation,pathology, trauma or any other event affecting theirintegrity has driven the search for rehabilitating ther-apies. Some of these include repair or replacementby means of autologous transplants, allogeneictransplants and more recently, tissue engineering,which integrates cell biology, molecular biology,genetics and biotechnology to produce complex tis-sues or organs. This study seeks to apply some ofthe basic principles of tissue engineering to obtain acomplex structure formed by tooth-like tissues anduse it in tooth regeneration trials.Tooth formation is the result of sequential, reiter-ative, and reciprocal interactions between ecto-dermal and mesenchymal tissues, through theexpression of signaling molecules families drivingtissues and the cells contained within along diversedifferentiation pathways1. A wide range of studiesat cellular and molecular levels have provided suf-ficient evidence to establish that the embryonic oralepithelium provides inductive signaling for toothinitiation. In the early stages of mandibular devel-opment in mice, all the ectomesenchymal cellsseem to be capable of responding to signals fromthe oral epithelium2. The ability of the embryonicoral epithelium in early developmental stages (com-petent dental epithelium) to direct odontogenesiswhen recombined with non-oral ectomesenchymesuggests that ectomesenchymal cells are plastic intheir responses to oral epithelial signals 3. The useof embryonic tissues as source of inductive signalsfor organogenesis and their subsequent transplanta-tion to receptor sites in adult individuals for thedevelopment of a complex multicellular structurein vivo, requires an animal model in which immu-nity due to histocompatibility does not pose anoth-er variable, therefore it is mandatory to use aninbred strain for this type of experiment. The Lewisinbred rat strain is one of the most frequently usedanimal models for transplant experiments4 due toits high level of homozygosity.Cellular proliferation and maturation during organo-genesis requires a support structure in order to facil-itate interactions, and in this regard the function ofthe scaffold has evolved into an instructive micro-environment facilitating cell adhesion, migration,differentiation and subsequently its own degrada-tion5. Synthetic polyethylene glycol diacrylate

(PEGDA) scaffolds are commonly used biomateri-als in tissue engineering because their chemical andphysical properties such as resistance to flexion, ten-sion, compression and shearing can easily be modi-fied according to the individual needs of thedeveloping tissue5,6. They are used as drug and celltransporters7, have been approved by the FDA (USFood and Drug Administration) for various medicalapplications6 and some are formed from a variety ofpolymers that are water-soluble or interact withwater, endowing them with high permeability8. Theycan be molded by photopolymerization processesgentle enough to be performed in presence of livingcells, which in turn can become homogeneously dis-tributed within the structure9. Polyethylene glycolbased hydrogels have proven to be biocompatible,and support the viability of cells encapsulated with-in them 10,11. Implantation of PEGDA constructs hasshown that the scaffold breaks down, allowing theformation of a cell matrix with the microscopicstructures distinctive of the different cell layersincluded in it12. The aim of this study was to com-bine rat embryonic oral ectodermal cells (withpotential to induce odontogenesis) and mesenchy-mal stem cells from adult rats in a hydrogel scaffoldand implant them in vivo to achieve the formationof a structure containing tissues of dental origin.

MATERIAL AND METHODS

Animals

This study followed the legal provisions of TheInternational Guiding Principles for BiomedicalResearch Involving Animals developed by theCIOMS (Council for International Organizations ofMedical Sciences), which includes ethical and legalaspects of experimenting with animal models. Thelaboratory procedures were performed according tothe standards for Biosafety for managing and dis-posing of biological specimens of the DentalResearch Center of the School of Dentistry, Pontif-icia Universidad Javeriana and the Center for Ani-mal Experiments of the School of Dentistry,Pontificia Universidad Javeriana. This Project wasapproved by the Ethics and Research Committee ofthe School of Dentistry, Pontificia UniversidadJaveriana as part of the project “In vivo Obtentionof a tooth-like structure from odontogenic rat cellsin a hydrogel scaffold”. Approximately ten 8-week-old sibling pairs ofmale (150-225g) and female (100-200g) Lewis

244 L. Jaramillo, et al.

Acta Odontol. Latinoam. 2012 ISSN 0326-4815 Vol. 25 Nº 3 / 2012 / 243-254


(LEW/ SsNHsd) rats were purchased from HarlanLaboratories, Inc. (Indianapolis, IN) and matedmonogamously to generate the foundation colony.The pairs were retired from the foundation colonyafter the third pregnancy and replaced with a newsibling pair. The offspring produced were used forbreeding either in the foundation colony or in theproduction colony used for experiments. Male andfemale rats in the production colony were group-housed in a One Cage® ventilated microisolator(Lab Products, Inc.) (four or five rats per cage) indisinfected polycarbonate cages (36 cm × 49 cm ×21.2 cm; One Cage, Lab Products, Inc., Seaford,DE) according to age and sex. For breeding, ratswere housed in pairs in polycarbonate cages (36cm × 23.5 cm × 21.5 cm; One Cage, Lab Products,Inc.). All cages were covered with filter tops andcontained sterile pine wood chips (Depósito deMaderas las Palmas SAS, Bogotá, Colombia). Thecubicle containing the microisolator had an aver-age of 50 filtered air (HEPA)turnovers/hour, tem-perature 22+/- 1°C, relative humidity 55 to 65%and 12:12 light/dark cycle (lights on at 06:30 a.m.and off at 6:30 p.m.). All animals were transferredto clean, disinfected cages with a fresh bed twice aweek. Sterilized feed (Rodentina Agrinal Colom-bia SA.) and water were provided ad libitum fromthe time of entry until euthanasia. Once a week,water was supplemented with a vitamin/amino-acid complex (Promocalier L, Laboratorios CalierSA.) to compensate for the nutrients lost throughfeed sterilization. Microbiological and serological monitoring wasperformed twice a year. The results were negativefor the following pathogens: Mycoplasma pulmo-nis, Sendai virus, CAR bacillus, Encephalitozooncuniculi, IDIR, Hantaan, LCMV, MAD1, MAD2,RPV, RMV, KRV, H1, PVM, RCV/SDAV, REO3,RTV and Clostridium piliforme. The rats werealso negative for Helicobacter spp., Pasteurellaspp., Streptobacillus moniliformis, Streptococcus(Streptoccoco β haemolitic group A), Salmonellaspp, Leisteria monocytogenes, Corynebacteriumkutscheri, Pneumococcis spp, Sarcoptes scabiei,Cestodes (intestinal parasites), Spironucleus muris(intestinal parasites), Giardia muris, Siphacia obve-lata, Aspiculuris tetraptera, all arthropods, allhelminthes and all intestinal protozoa, therefore thehealth of the individuals housed at the SEA FOUJqualified as SPF (Specific Pathogen Free).

Population

34 pregnant females (10 - 38 weeks old) were used asa source of embryonic tissues, 70 males (10 - 38 weeksold) as implant recipients and 5 females (10 - 21 weeksold) as bone marrow donors (109 animals altogether).

Humane euthanizing conditions

All individuals were euthanized by asphyxia with amixture of CO2/air followed by cervical dislocation.

Culture conditions

Whenever culture medium is mentioned, it refers toα-MEM (Gibco 12000-022) supplemented with10% bovine fetal serum (Gibco 16000-044) and 1%penicillin /streptomycin /amphotericin B (Gibco15240-062). All incubation steps were performedat 37ºC and 5% CO2, and the culture medium waschanged every two days. In vitro culture time forthe constructs was 48 to 72 hours.

Bone marrow stem cell culture

The aseptic dissection technique for lower limbbones (tibia and femur) of humanely euthanizedyoung females to obtain bone marrow stem cells wasstandardized. The marrow was flushed using asyringe with an 18-gauge needle loaded with 1 mlof culture medium, mechanically disaggregatedusing the same needles, and centrifuged at 2600xgfor five minutes. The supernatant was discarded, theprecipitate re-suspended in culture medium andseeded in 25 cm2 cell culture dishes to establish theprimary culture. When 80-90% cell confluence wasreached, cells were detached by incubation with 1ml trypsin-EDTA solution (Sigma T-4049), collect-ed by centrifuging, and transferred to fresh vials at adensity of 5-7 x 105 cells/vial, according to theNeubauer chamber count using the 0.4% TrypanBlue (Sigma T-8154) exclusion principle. Con-structs were produced using the detached cells whenthe first passage was confluent (second passage).

Embryo production

In order to perform controlled crosses, estrus wasdetermined in in the production colony females bymeasuring the vaginal wall impedance using anestrus cycle monitor (EC-40 Estrus Cycle Monitor®,Fine Science Tools). Females in estrus were matedduring the night with trained males, separated thenext day and the service was confirmed by evaluat-ing the presence of a vaginal plug or sperm in vagi-

Odontogenic Cell Culture in PEGDA Hydrogel 245



nal lavage, in which case the female was consid-ered to be pregnant and day zero for embryo devel-opment was set. The time to harvest embryos was determined byevaluating the concurrent expression of three puta-tive genes, namely Pitx2, Shh and Wnt10a, with thewhole-mount in situ hybridization technique atE11.5, E12, E12.5, E13 and E13.5 using RNAprobes (sense and anti-sense). The probes weremade from plasmids for each target gene donatedby Dr. Irma Thesleff of the Institute of Biotechnol-ogy, University of Helsinki. Briefly, the eluted plas-mid was amplified by cloning using an E. colitransforming strain, purified and linearized usingrestriction enzymes. The linearized plasmid waspurified again, transcribed to obtain the RNAprobes, which were applied to the embryos to eval-uate the expression of the target genes. On the day set for embryo harvesting, pregnantfemales were humanely euthanized and a midven-tral laparotomy was used to expose and remove theuterine horns which were placed in PBS with calci-um and magnesium (DPBS) (Sigma D-1283).Embryos were removed from the yolk sac andplaced in clean DPBS solution.

Embryonic ectoderm tissue preparation

Using hypodermic needles under a stereomicro-scope (Nikon SMZ 1500), the mandibular segmentof the first branchial arch was dissected and divid-ed into two halves. The posterior segment of eachof the two halves was again dissected, separating

the portion containing the competent embryonicoral ectoderm (EOE) (Fig. 1 a and b). The dissectedfragments were treated with 1 ml of trypsin-pancre-atin solution (Sigma T-4799-Sigma P-3292) andincubated for 20 minutes at 37ºC to separate theectodermal layer from the mesenchymal tissue (Fig.1 c). After enzymatic digestion, the EOE fragmentswere incubated in culture medium at 37ºC until thethree-dimensional constructs were prepared, proce-dure that was completed within the hour followingeuthanasia (Fig. 1 d).

Construct preparation

Three groups of constructs were prepared: experi-mental (seeded with BMSC y el EOE), negativecontrol (seeded only with BMSC) and positive con-trol (seeded with tooth germ ectodermal and mes-enchymal cells at E12.5, E19 and 4PN). The PEGDA was prepared at a concentration of10% in PBS supplemented with a 1% antibiotic andantimycotic solution, to which a 0.1% solution ofphotoinitiator (Ciba, Irgacure 2959) was added.Polymerization was achieved by exposure to a365nm UV radiation (Spectroline®BIB-150P lamp),in a polypropylene (PP) pyramidal mold maintainedin an inert atmosphere (N2 flow) at a distance of15cm. Temperature of the mold was kept at 37ºC.Hydrogel loading capacity was tested by adding200,000; 400,000 and 700,000 cells per construct,finding that full polymerization was achieved when400,000 cells were included in 25 μl of total hydro-gel volume. After polymerization the construct was



Fig. 1: Embryo dissection andectoderm separation. a) Embryoharvest at E12.5. b) Dissected frag-ments are stored in PBS with Caand Mg. c) Enzymatic separation oftissues from fragments in a pancre-atin/trypsin solution, the ectodermseparates cleanly from the mes-enchymal portion of the fragment.d) Competent ectoderm fragmentused in experiments. Scale bars: a) 5mm, b) 300µm, c and d) 125µm.


removed from the mold and cultured in vitro untilimplantation. Most of the constructs were implant-ed, but some were used for preimplantation histo-logical analysis and viability tests. Cell viabilitywithin the construct was initially assessed by meansof histological analysis and confirmed by Promegalive and dead cell staining (Invitrogen Live/Dead,L3224), the results of which were viewed under flu-orescence microscope (Fig. 2).

Experimental constructs

Constructs were prepared in two phases, ectoder-mal and mesenchymal, which were separately poly-merized within the mold. The ectodermal phase wasconstructed by loading 5 µl of the hydrogel solu-tion containing EOE fragments (5 to 15) in the apexof the pyramidal mold. The mesenchymal phasewas prepared by diluting 400,000 cells in 25µlhydrogel, pouring it over the ectodermal phase andpolymerizing.

Positive Controls

Three types of positive controls were used. Thetooth germ was dissected at the same gestationalage as the experimental constructs and ectodermand mesenchyme were separated. The mesenchymewas disaggregated with a dipase solution (Gibco17105041) in PBS 2U/ml, incubated for 20 minutesand then used to produce the constructs by meansof the process described above for preparing exper-imental constructs. A second type of positive con-trol was prepared by dissecting the tooth germs at a

more advanced gestational stage (E19.5), andincluding the dissected germ without disaggregat-ing in PEGDA hydrogel. The third type of positivecontrol was constructed by dissecting neonate toothgerms (4PN), separating the ectoderm from themesenchyme by mechanical dissection, and re-aggregating it in a hydrogel mold.

Negative Controls

Negative controls were prepared by including400,000 BMSC in PEGDA.

Implantation surgery

Implantation surgery was performed under sterileconditions in the operating room. The animal wasplaced in an induction chamber and anesthetized(Ohmeda vaporizer model Fluotec 4 using 3%Halothane with an oxygen flow between 1.5 and 2l/min. After induction, the abdomen was shaved anddisinfected with 0.2% clorhexidine solution. The ani-mal was reintroduced in the induction chamber andafterwards connected by means of a rodent mask toan open anesthesia circuit and maintained with 1.5-2% Isoflurane (FORENE®) (Ohmeda vaporizermodel Isotec 3) and a 1.5-2 l/min oxygen flow. Heartrate and oxygen saturation were monitored (OhmedaBiox 3700 pulsoximeter®) with the pulsoximeter’sprobe (Ohmeda Veterinary Pulse Oximeter LingualSensor®) attached to the animal’s tail. Heart rate wasmaintained at about 250 bpm and saturation over90% during the 25-minute surgery. At normal oper-ating room ambient temperature (21C), small rodents



Fig. 2: Determination of pre-implan-tation construct cell viability usingPromega’s live and dead cell stain.a) Control with live cells. b) Controlwith dead cells. c) Mesenchymalcells seeded in a three-dimensionalhydrogel (PEGDA) scaffold. Scalebars: a and c) 30µm, b) 60 µm.


can rapidly lose body temperature due to heat dissi-pation, therefore a thermic blanket was placed on topof the surgical table to avoid hypothermia during thesurgical procedure. To gain access to the implantation site the animal wasplaced in a decubitus supine position and a 1.5 cmmidventral incision was performed over the LineaAlba 3 cm caudal of the xiphoid appendix. A layer dis-section was performed until exposure of the parietalperitoneum, which was then cut in order to gain accessto the peritoneal cavity. The peritoneum was cut andtissue forceps used to extract the ileal portion of thesmall intestine, which was extended on the shavedabdominal region. For implantation, two triangularareas of mesentery delineated by nutrient vessels andthe intestine wall were selected (Fig. 3a), upon whicha casing was made by passing a continuous 6-0 suturethrough the perivascular fat flanking the selected area(Fig. 3b), taking care not to strangle the vessels orbreak the mesentery or the construct upon closing(Figs. 3c and d). The ileum was replaced inside theabdominal cavity, the muscular and fascial layersclosed in one plain with a simple continuous suture,and the skin with simple interrupted sutures both using4-0 silk. Postsurgical analgesia was achieved by oraladministration of a 0.62% morphine/water solution(2.5 mg/kg) dosed every 6 hours for 24 hours. Oncethe constructs had reached the selected maturationtime (4-5 weeks), the animals were euthanized and theconstructs recovered and fixed in a buffered formalinsolution (Sigma HT501528), after which they wereprocessed and stained with hematoxylin eosin (HE).

RESULTS

In this study, couplings to obtain embryos needed for theexperiments were scheduled by estrus determination bymeans of vaginal wall impedance readings, which showedthat 16.75% of the females sampled were in estrus, 45%were served and 27.7% carried embryos upon sacrifice. Atotal 34 pregnant females were euthanized humanely, fromwhich an average 7.2 embryos/female were obtained.Embryo development was not necessarily homogeneous,and sometimes differed by up to 0.5 days. Only EOE fromembryos with uniform development was used for prepar-ing the constructs. The whole-mount in situ hybridization techniqueshowed that the expression of selected tooth initiationmarkers (Pitx2 Shh and Wnt10a) in EOE is diffuse orhas concentration gradients at stages E11.5 and E12,and is more localized to the areas of future dentaldevelopment during gestational stages 12.5 and E13.5.Prior to this stage, the signal is distributed diffusely inthe first and second branchial arches ectodermal tissues. Similarly, during the development stagesobserved, the Shh and Pitx2 signals, particularly theformer, are located in the limb formation control cen-ters (ZPA) appearing first in the front limbs (E11.5)and later in the hind limbs (E13.5). The concentratedexpression of these three genes was indicative of theappearance of tooth initiation signals, defining themoment for harvesting embryos to be used in constructpreparation. The dissection of the mandibular segmentof the first branchial arch and its subsequent enzymat-ic treatment allowed ectodermal and mesenchymalportions to be separated, and the EOE was used in the



Fig. 3: Construct implantation. a) The terminal mesenteric vessels(arteries and veins) fan out and withthe wall of the small intestine delimittriangles of mesenterium. b) A con-tinuous 6-0 suture was passedthrough the perivascular fat adjacentflanking the triangle and forming thecasing. Extreme care was taken sothat upon closing the suture wouldnot strangle the nutrient vessels. c) Closed casing containing the con-struct. d) Construct in positionsurrounded by mesentery and nutri-ent vessels.


experiments to induce tooth differentiation. The inclu-sion of these fragments as a group in a small volumeof hydrogel formed the ectodermal phase of the con-structs, which was subsequently attached to the mes-enchymal phase, constituted by bone marrow purifiedcells cultures used as initiation signal receptors. Purification of the sub-population of stem cells wasbased on their ability to adhere to the surfaces onwhich they were seeded, expanding in two directions,and hematopoietic cells were progressively excludedby means of successive medium changes. A homoge-neous cell population was obtained at second passage,and at this stage they were used to produce the three-dimensional constructs. Including 400,000 BMSC’sin a minimum hydrogel volume of 25 μl allowed theproduction of a three-dimensional structure with nopolymerization defects and easy to manipulate duringthe in vitro culture and in vivo implantation processes.Figure 2 shows live cells remaining in PEGDA con-structs after three days’ culture in vitro. These resultsguided the production and implantation of 76 con-structs; 37 experimental (17 at E12.5 and 20 at E13),12 positive controls and 27 negative controls, in theileal mesentery where they matured for 3 to 5 weeks

surrounded by vessels and perivascular fat. The select-ed implantation site facilitated the recovery of the con-structs and dissection of the surrounding tissues. Histological analysis of the experimental controlsshowed that the EOE remained viable and proliferat-ed, becoming invaginated within the hydrogel, whilethe mesenchymal cells did not. In order to enablecloser contact among BMSC population in anattempt to stimulate them to maintain viability, a hol-low pyramidal PEGDA mold was designed (Fig. 4a)that allowed for condensation of the BMSCs over theectoderm fragments by means of a brief centrifuga-tion prior to hydrogel polymerization. The constructsthus formed were maintained in culture for 7 days(Fig. 4 b), and then observed using an inverted micro-scope. Contraction of the cell mass towards the baseof the pyramid was detected (Fig. 4c). Although con-centration of BMSCs was increased, thus increasingcell density in the hydrogel/cell solution before poly-merization, results did not improve cell viability inthe implanted constructs (Fig. 4d and e). Non-prolif-eration of BMSCs in PEGDA was confirmed withresults of the negative controls, all of which showedthe BMSCs undergoing cell death (Fig. 4f).



Fig. 4: PEGDA constructs. a) Hollow pyramidal mold. This hollow mold allowed BMSCs to be condensed over ectodermal cells withgentle centrifugation before polymerizing the hydrogel. b) Pyramid with hydrogel loaded with BMSC and polymerized inside the hol-low mold, after being cultured in α-MEM for 7 days, there is a contraction in cell mass towards the base of the pyramid. c) Invertedmicroscope image of the BMSC culture embedded in hydrogel; picture taken of the pyramid in Figure b. d) Histological section of theBMSC from the previous picture stained with hematoxylin eosin (HE), cells show all phases of anoikis (karyopyknosis, karyorrhexisand karyolysis) e) Section of an experimental construct recovered at 5 weeks, showing the vital proliferative ectoderm. The mes-enchymal portion of the construct is occupied by cell remains. f) Section of a negative control recovered at 5 weeks, picture of themesenchymal portion similar to the one in e, HE stain. Scale bars a and b) 1 mm, c and e) 50µm, d) 25 µm and f )150 µm.


In some complete experiments, where the epithelialportion was in direct contact with the mesentery, anexternal vascular network was created (Fig. 5a)extending into the ectodermal portion, and somecells of the mesenterium acquired a columnar mor-phology similar to that of odontoblasts at the begin-ning of dentin formation (Fig. 5b).In the positive controls, histological sections showthat the hydrogel allows nutrient diffusion but pre-vents the vascular proliferation that greatly con-tributes to final tooth development. Vascularproliferation only occurred in cases in which therewas a gap in the hydrogel cover as a result of con-struct breakage at the time of implantation.In the (E19.5) positive controls, tooth germs wereincluded within the PEGDA (Fig 6a) but there was

involution of the odontogenic complex after implan-tation, leading to the formation of a epidermal inclu-sion cyst-like structure (Fig 6b) with an ectodermalportion of keratinized stratified squamous epitheliumand a defined lumen containing shedded keratin (Fig.6c) and (Fig. 6d). At E12.5, when the posterior seg-ment of the mandibular portion of the first branchialarch was embedded in hydrogel and implanted, a cir-cular primordium formed with stellate reticulum-likecell structure surrounded by ectodermal columnarcells, considered to be nodular structures predeces-sors of dental structures (Fig. 6e). When a four-daypost-natal (PN4) germ was used as positive control,interaction was achieved between BMSCs and EOE,forming a structure with dental tissues but lackingclearly defined organization (Fig. 6f).



Fig. 5: Experimental construct at 5weeks’ implantation. a) View of anexternal vascular network associatedwith the epithelial portion in direct con-tact with the mesenteric membrane. b)The vascular contribution is distributedwithin the ectoderm. The mesenterycells in contact with the embryonicectoderm acquire a columnar morphol-ogy, HE stain.

Fig. 6: Positive controls. a) Macroscopic image of a positive control of an E19.5 tooth germ embedded in hydrogel and implanted inthe mesentery for 5 weeks. b, c and d) Histological images of the specimen showing a layer of keratinized stratified squamous epithe-lium. The lumen is filled with sloughed keratin, HE stain. e) Nodular structures which are precursors of tooth structures in a positivecontrol made with an E12.5 germ, implanted for 5 weeks. A circular primordium can be seen, which contains a stellate reticulum-likecell structure surrounded by columnar ectodermal cells. f) Positive control with a 4-day post-natal (PN4) germ. Deposition of irreg-ularly shaped matrix of enamel and pre-dentin. Scale bars: a) 500µm, b) 300µm, c) 100µm, d and e) 30µm, f) 60µm.


DISCUSSION

Tooth production by means of tissue engineering isan important aim in dental research, which is whythis study seeks to establish three-dimensionalmicro-environments establishing communicationpathways among different cell types. Those interac-tions would be homologous to the ones occurringnaturally during tooth development, enabling theregeneration of dental tissues. A tooth is the resultof the differentiation of epithelial cells intoameloblasts and of ectomesenchymal cells intoodontoblasts. These differentiated cells produceenamel and dentin matrix13 through sequential, reit-erative, and reciprocal interactions of signaling mol-ecules between the ectodermal and mesenchymaltissues1. Induction of the signaling cascade usingcompetent embryonic ectoderm and mesenchymaltissue of non-dental origin has been extensivelydescribed14, demonstrating that in early stages ofmouse development, the epithelium covering thestomodeum of the mandibular portion of the firstbranchial arch provides inductive signals for toothmorphogenesis and histodifferentiation to non-den-tal origin mesenchyme suggesting that ectomes-enchymal cells are plastic in their responses tosignals form the oral epithelium. There are two mainapproaches to tooth regeneration; the first based ontissue engineering, which seeks to regenerate teethby seeding disaggregated tooth germ cells on bio-materials that serve as scaffolds, while the secondinvolves reproducing the development processes intooth formation using embryonic dental tissues asinductors15. This study combined the two approach-es by seeding embryonic cells and stem cells fromadult individuals in a PEGDA scaffold which wassubsequently implanted in vivo in rats. PEGDAhydrogel was selected as the scaffold because priorresearch has enabled the in vivo production of astructure in the shape of a human mandibularcondyle from a population of mesenchymal stemcells induced to differentiate into chondrogenic andosteogenic lineages12 allowing stratification ofembryonic ectodermal tissues and mesenchymalBMSC by polymerizing separate layers which main-tain the assigned spatial relation. This property wasthe main reason why PEGDA hydrogel was selectedas the scaffold for producing the constructs. In order to obtain the 234 embryos used, scheduledmatings were performed using females that testedpositive for estrus by means of vaginal wall imped-

ance reading, improving the service/pregnancy ratioand contributing to the application of the 3Rs prin-ciple in animal model research, since by reducingpseudo-pregnancies the number of available femalesfor periodical sampling remained stable16. Different studies have used mouse competent embry-onic dental ectoderm in tissue engineering experi-ments, to induce differentiation in adult bonemarrow-derived stem cells, achieving in vivo forma-tion of complex dental structures17, 18. Collectingectodermal cells from rat embryos offered advan-tages because due to their size, the cell volume har-vested is greater, tissue manipulation during themicro-dissection procedures is easier and it facili-tates future implantation of the germs obtained bycell recombination in individuals of the same species.However, there is little information in the literatureregarding embryonic tooth development and theappearance of putative initiation signals for the for-mation of teeth in rats, which is why it was neces-sary to use the whole-mount in situ hybridizationtechnique to evaluate the location of the expressionof three putative signaling molecules in embryonicoral ectoderm associated with dental competence. InLewis rat embryos at different stages of development,the results showed that there is a delay of approxi-mately two days in the appearance of initiation sig-nals compared to the information reported for mice.While in mice, the initiation signals, especially theexpression of Wnt10a and Pitx2 are concentrated inthe embryonic ectoderm that covers the stomodeumat E10.5, in rats this concentration is expressed atE12.5. This is why the ectoderm covering the sto-modeum of the mandibular processes of the firstbranchial arch in Lewis rat embryos, between E12.5and E13.5, is competent for inducing in the underly-ing mesenchyme the signaling cascade needed forthe formation of dental structures. Careful schedul-ing was needed to coordinate the time at which ecto-dermal tissue was harvested and the progress of themesenchymal cultures because as from the date ofthe scheduled crossing, there were 12.5 to 13.5 daysto have the BMSCs in second passage. The implant sites reported in other studies wereselected based on criteria of immunoprivilege, sur-gical approach, presence of nutrient contribution andease of implant recovery. The most frequently citedsites include kidney subcapsular space, grater omen-tum, intraoclular space and subdermal pockets19,20.In this study mesentery was selected as the construct




receptor site because the surgical approach is sim-pler, it has plentiful nutrient contribution and con-struct recovery is easier, and the use of subcapsularspace avoided because it limits the size of theimplants that can be placed and also because in thepresent study immunoprivilege did not play animportant role, considering that an isogenic strainwas used both as source of tissues and constructreceptor. Previous studies have reported the use ofextracellular matrix (ECM) from decellularizedmesenteric membrane as scaffold for cell inclusionin tissue engineering. The ECM is a complex net-work of collagen, fibronectin and other proteinsintermeshed with proteoglycans21 which serves notonly as support material but also regulates cell func-tions such cell proliferation, migration and differen-tiation22 and modulates the transduction of signalsactivated by various bioactive molecules such asgrowth factors and cytokines23. Reports publishedafter the initiation of this study, mention the mesen-terium as an appropriate receptor site for implantingislets of Langerhans in experiments pursuing recov-ery of pancreatic function in animals affected by dia-betes mellitus24. Other study reported implantationof hepatocyte progenitor cells in the mesentericleaves of animals, enabling them to proliferate andfill the spaces of the macroporous structure, suggest-ing the feasibility of establishing three-dimensionalcultures of hepatocyte progenitor cells for hepatictherapies based on tissue engineering25. The histological evaluation of the implantation ofexperimental, positive controls and negative controlimplants shows that the selected site facilitates cellinteractions by providing nutrients that ensure cellproliferation, and sometimes, when the ectodermalcells in the construct made contact with the mesen-tery, complex multicellular structures were formedwith a well-developed vascular network. The EOEincluded in the constructs remained viable through-out the implantation period but in spite of the factthat the hydrogel allows the proliferation of differen-tiated cells, its physicochemical characteristics pro-mote stem cell death26. In some specimens and as aresult of cell degradation, this produced cystic spacessurrounded by chronic inflammatory infiltrate. Thehistological analysis of the negative controls showedthat in all implanted constructs, the BMSCs under-went a process of cell death characterized by kary-opyknosis, karyorrhexis and karyolysis, which wasreported after the completion of the experimental

phase of this project26 and has been called anoikis,(apoptosis induced by lack of interaction with pro-teins of the extracellular matrix). In all the negativecontrols that were recovered, the BMSC site showedno vascularization, suggesting that the hydrogel mayact as a barrier that allows nutrient diffusion but atthe same time prevents vascularization, for whichdirect contact between the mesenteric membrane andimplanted cells is needed. A different situation was observed in the positivecontrols because the germ included in the hydrogelwas made up of mesenchymal tissue with matureextracellular matrix supporting the cells. At (E19.5)there was involution of the odontogenic complexleading to the formation of a epidermal inclusioncyst-like structure with a defined lumen containingsloughed keratin and an ectodermal lining of kera-tinized stratified squamous epithelium, indicatingthat further development of the cultured germ doesnot ensure completion of morphogenesis and his-todifferentiation processes. The timing of germ har-vesting and the presence of small quantities ofnon-dental tissue may contribute to this situation,as previously reported27. At PN4, cell interactionwas achieved and a structure containing dental tis-sues lacking clear organization was formed. Final-ly, histological assessment of the experimentalconstructs showed that EOE remained viable andproliferated, invaginating in the hydrogel. Howev-er, as in the negative controls, the mesenchymalcells underwent anoikis, which was the determin-ing factor in the absence of dental tissue formationwhen the two cell types were included in a PEGDAhydrogel matrix. A frequent histological finding inthe experimental constructs shows that mesenchy-mal mesenteric cells in contact with the embryonicectoderm acquired a columnar morphology similarto odontoblasts at the beginning of dentin forma-tion. This may be related to the ability of the mesen-tery ECM to foster mesenchymal cell proliferationand differentiation when it comes into contact withEOE, by contributing growth factors, cytokines andvascularization to the tissue. Nodular structures of ectodermal origin described asdental structure precursors28 were often found in theE12.5 positive controls. This demonstrates that undercertain circumstances there may be cell interactionsinside the scaffold, but they stop before dental tis-sues differentiation at the selected implantationtimes. Explanations on why this happens can relate




to the fact that initiation signals at the selected har-vesting stage are not strong enough or the EOE cellvolume included in the scaffold is deficient and bothimply insufficient intensity of initiation signals nec-essary to induce differentiation in the BMSCs27.Finally, during BMSC culture, it might be necessaryto supplement the medium with growth factors andcytokines related to mesenchymal competence (sig-naling molecules that control gene expression in themesenchyme) and which would complete or rein-force the signals required for differentiation towardsthe dental lineage. The use of other types of support-ing matrices such as collagen gel or sponges to sup-port tooth germ cells dissociated in later developmentstages has been reported, and they have been shown

to foster cell interactions. Their use in the case ofearly interactions such as those that occur betweenthe BMSC and EOE should be examined in order toachieve an ideal dental primordium.

CONCLUSION

PEGDA hydrogel supports the viability and prolifer-ation of EOE but not of BMSC. The latter undergoapoptosis, preventing the existence of a cell groupthat could act as a receptor for the initiation signalsoriginated in the ectoderm necessary for tooth for-mation. The search must continue for an ideal sup-port that will maintain the three-dimensionalstratification of cells, enable their manipulation dur-ing implantation, and facilitate vascular infiltration.



ACKNOWLEDGMENTS

This study was funded by Colciencias and the Academic ViceRector’s Office of the Pontificia Universidad Javeriana. Theauthors would like to thank Dr. María Beatriz Ferro, AcademicDean of the School of Dentistry of the Pontificia UniversidadJaveriana for her invaluable support in conducting this study, Dr.Irma Thesleff for her generous donation of the plamids and fortraining the researchers in techniques necessary for the develop-ment of this project and, DICHCOL SAS. for their contributionto the special sterilization protocols used for this study and Mrs.Olga Lucía Beltrán for her conscientious care of the colony.

CORRESPONDENCE

Dr. Camilo DuránCra. 7 No. 40-62 Edificio 25 Piso 4 Bogotá D.C, ColombiaEmail: [email protected]

REFERENCES1. Thesleff I, Tummers M. Tooth organogenesis and regenera-

tion. Harvard Stem Cell Institute. Cambridge (MA), 2009. 2. Ohazama A, Modino SA, Miletich I, Sharpe PT. Stem-cell

based tissue engineering of murine teeth. J Dent Res 2004;83:518-522.

3. Matalova E, Tucker AS, Sharpe PT. Death in the life of atooth. J Dent Res 2004; 83:11-16.

4. Modi HN, Suh SW, Prjvc B, Hong JY, Yang JH, Park YH,Lee JM, Kwon YH. Bone quality and growth characteristicsof growth plates following limb transplantation between ani-mals of different ages—results of an experimental study inmale syngeneic rats. J Orthop Surg Res 2011;6:53.

5. Bahney CS, Lujan TJ, Hsu CW, Bottlang M, West JL, John-stone B. Visible light photoinitiation of mesenchymal stemcell-laden bioresponsive hydrogels. Eur Cell Mater 2011;15:43-55.

6. Durst CA, Cuchiara MP, Mansfield EG, West JL, Grande-Allen KJ. Flexural characterization of cell encapsulatedPEGDA hydrogels with applications for tissue engineeredheart valves. Acta Biomater 2011;7:2467-2476.

7. Hoffman AS. Hydrogels for biomedical applications. AdvDrug Deliv Rev 2002;54:3-12.

8. Nguyen KT, West JL. Photopolymerizable hydrogels for tissue engineering applications. Biomaterials 2002;23:4307-4314.

9. Ouasti S, Donno R, Cellesi F, Sherratt MJ, Terenghi G,Tirelli N. Network connectivity, mechanical properties andcell adhesion for hyaluronic acid/PEG hydrogels. Biomate-rials 2011;32:6456-6470.

10. Poshusta AK, Anseth KS. Photopolymerized biomaterialsfor application in the temporomandibular joint. Cells Tis-sues Organs 2001;169:272-278.

11. Burdick JA, Mason MN, Hinman AD, Thorne K, AnsethKS. Delivery of osteoinductive growth factors from degrad-able PEG hydrogels influences osteoblast differentiationand mineralization. J Control Release 2002;83:53-63.

12. Alhadlaq A, Mao JJ. Tissue-engineered neogenesis ofhuman-shaped mandibular condyle from rat mesenchymalstem cells. J Dent Res 2003;82:951-956.

13. Honda MJ, Ohara T, Sumita Y, Ogaeri T, Kagami H, UedaM. Preliminary study of tissue-engineered odontogenesisin the canine jaw. J Oral Maxillofac Surg 2006;64:283-289.

14. Thesleff I, Vaahtokari A, Partanen AM. Regulation oforganogenesis. Common molecular mechanisms regulatingthe development of teeth and other organs. Int J Dev Biol1995;39:35-50.

15. Nakahara T, Ide Y. Tooth regeneration: implications for theuse of bioengineered organs in first-wave organ replace-ment. Hum Cell 2007;20:63-70.

16. Jaramillo L. Briceño I. Durán C. Optimization of pairing/ser-vice in a Lewis inbred rat colony by using vaginal imped-


ance for estrous cycle stage determination. Lab Anim 2012(in press).

17. Hu B, Nadiri A, Kuchler-Bopp S, Perrin-Schmitt F, PetersH, Lesot H. Tissue engineering of tooth crown, root, andperiodontium. Tissue Eng 2006;12:2069-2075.

18. Hu B, Unda F, Bopp-Kuchler S et al Bone marrow cells can give rise to ameloblast-like cells. J Dent Res 2006; 85:416-421.

19. Taylor AW. Ocular immune privilege. Eye (Lond) 2009;23:1885-1889.

20. Ng TF, Osawa H, Hori J, Young MJ, Streilein JW. Allogene-ic neonatal neuronal retina grafts display partial immuneprivilege in the subcapsular space of the kidney. J Immunol2002;169:5601-5606.

21. Lu H, Hoshiba T, Kawazoe N, Koda I, Song M, Chen G.Cultured cell-derived extracellular matrix scaffolds for tis-sue engineering. Biomaterials 2011;32:9658-9666.

22. Manabe R, Tsutsui K, Yamada T, Kimura M, Nakano I, Shi-mono C, Sanzen N, Furutani Y, Fukuda T, Oguri Y, Shimamo-to K, Kiyozumi D, Sato Y, Sado Y, Senoo H, Yamashina S,Fukuda S, Kawai J, Sugiura N, Kimata K, Hayashizaki Y,Sekiguchi K. Transcriptome-based systematic identification

of extracellular matrix proteins. Proc Natl Acad Sci USA2008;105:12849-12854.

23. Hynes RO. The extracellular matrix: not just pretty fibrils.Science 2009;326:1216-1219.

24. Rogers SA, Tripathi P, Mohanakumar T, Liapis H, Chen F,Talcott MR, Faulkner C, Hammerman MR. Engraftment ofcells from porcine islets of Langerhans following transplan-tation of pig pancreatic primordia in non-immunosuppresseddiabetic rhesus macaques. Organogenesis 2011; 7:154-162.

25. Sakai Y, Nishikawa M, Evenou F, Hamon M, Huang H, Mon-tagne KP, Kojima N, Fujii T, Niino T. Engineering of implantableliver tissues. Methods Mol Biol 2012; 826:189-216.

26. Salinas CN, Anseth KS. Mesenchymal stem cells for cran-iofacial tissue regeneration: designing hydrogel deliveryvehicles. J Dent Res. 2009;88:681-692.

27. Nakao K, Morita R, Saji Y, Ishida K, Tomita Y, Ogawa M,Saitoh M, Tomooka Y, Tsuji T. The development of a bioengi-neered organ germ method. Nat Methods 2007;4:227-230.

28. Sumita Y, Honda MJ, Ohara T, Tsuchiya S, Sagara H, Kaga-mi H, Ueda M. Performance of collagen sponge as a 3-Dscaffold for tooth-tissue engineering. Biomaterials 2006;27:3238-3248.




ODONTOGENIC CELL CULTURE IN PEGDA HYDROGEL...

Documents

Transcript of ODONTOGENIC CELL CULTURE IN PEGDA HYDROGEL...