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A Comparison of Bovine Bone and Hydroxyapatite Scaffolds During Initial Bone Regeneration: An In Vitro Evaluation Filiberto Mastrangelo, MD, DDS, PhD,* Raimondo Quaresima, BEng,† Alfredo Grilli, MD,‡ Lucia Tettamanti, MD,§ Raffaele Vinci, MD,k Gilberto Sammartino, MD, DDS, PhD,¶ Stefano Tetè, MD, DDS, PhD,# and Enrico Gherlone, MD, DDS**
n modern implantology, worldwide attention has been placed on the macroscopic implant design and surface characteristics to obtain rapid and predictable results in osteointegration.1–3 However, the success of simple and complex rehabilitation depends on implant placement in adequate quality and quantity of bone.4,5 Moreover, it is not always possible to find the jawbone of the implant suitable for proper implant rehabilitation due to the loss of 1 or more teeth.6 The alveolar bone resorption and remodeling processes produced a rapid loss of the normal maxillary or jawbone architecture.7,8 Additional surgical technique to current clinical success has evolved including
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*Young Researcher, Department of Oral Medical Science and Biotechnology, University of Chieti, Chieti, Italy. †Associate Professor of Biomaterial Engineering, Department of Civil Engineering, Architecture and Environment, University of L’Aquila, L’Aquila, Italy. ‡Ordinary Professor of Biology, Department of Oral Medical Science and Biotechnology, University of Chieti, Chieti, Italy; Leonardo da Vinci Telematic University, Torrevecchia Teatina (Chieti), Chieti, Italy. §Researcher of Pedodontics and Orthodontics, Department of Oral Science, Insubria University of Varese, Varese, Italy. kMaxillofacial Surgeon, Department of Oral Science, University Vita e Salute Milano, Milan, Italy. ¶Associate Professor of Maxillofacial Surgery, Department of Surgical Science, University Federico II Napoli, Naples, Italy. #Associate Professor of Oral Surgery, Department of Oral Medical Science and Biotechnology, University of Chieti, Chieti, Italy. **Ordinary Professor and Dean of Oral and Maxillofacial Surgery, Department of Oral Science, University Vita e Salute Milano, Milan, Italy.
Reprint requests and correspondence to: Mastrangelo Filiberto, MD, DDS, PhD, Department of Oral Science, University “G. d’Annunzio”, Chieti, Via dei Vestini 31, 66013 Chieti Scalo (CH), Italy, Phone: +39-3358390720, Fax: +39-0871-3554073, E-mail: fi[email protected] ISSN 1056-6163/13/02206-613 Implant Dentistry Volume 22 Number 6 Copyright © 2013 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0b013e3182a69858
Objectives: To evaluate the different behavior of 3-dimensional biomaterial scaffoldsdBovine Bone (BB; Bio-Oss) and Hydroxyapatite (HA; ENGIpore)dduring initial bone healing and development. Materials and Methods: Human dental papilla stem cells (hDPaSCs) were selected with FACsorter cytofluorimetric analysis, cultured with osteogenic medium, and analyzed with Alizarin red stained after differentiation. The obtained osteoblast-like cells (OCs) were cultured with BB and HA. alkaline phosphatase (ALP), OC, MEPE, and runt-related transcription factor 2 (RUNX2) expression markers were investigated performing Western blot and reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. After 40 days, samples were analyzed by light and electron microscopy.
Results: All the samples showed high in vitro biocompatibility and qualitative differences of OCs adhesion. RT-PCR and Western blot data exhibited similar marker rate, but ALP, OC, MEPE, and RUNX2expression, during initial healing and bone regeneration phase, was higher and faster in human dental papilla onto BB than in HA scaffolds. In biomaterials growth, RUNX2 seems to play an important role as a key regulator in human OCs from dental papilla bone development. Conclusion: Different surface BB scaffold characteristics seem to play a critical role in OCs differentiation showing different time of bone regeneration morphological characteristics as well as higher and faster levels of all observed markers. (Implant Dent 2013;22:613–622) Key Words: Bio-Oss, ENGIpore, bone, regeneration, osteogenesis, mesenchymal stem cells
the use of autologous or heterologous bone grafts to obtain bone regeneration and dental implant osteointegration.9,10 Several clinical studies have been performed to evaluate the use of different autologous or heterologous biomaterials scaffolds.11–13 Recent advances in clinical biotechnology research found interesting results of tissue regeneration, and several
reviews have critically analyzed the histological and clinical effects.12,13 Novel strategies for bone loss regeneration focus on tissue engineering, combine cell biology, and biomedical engineering, including stem cells and progenitor cells, bioactive scaffolds, and growth factors.14–16 Different biomaterials have been proposed and used for bone grafting: intraoral17,18 or
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extraoral19,20 autologous bone, allogenic grafts,21 xenografts,22,23 alloplastic grafts,24,25 or a combination of the same.26,27 The integration of these materials with the host tissue occurs by osteogenesis, osteoconduction, and osteoinduction.28 The main clinical advantage of bone regeneration with heterologous grafts was linked to the characteristics of the materials and the facilities to obtaining large quantities of materials.29 However, the different behavior between in vitro and in vivo tissue engineering development needs to be investigated as to progenitor cell biology, cell-to-cell interactions, molecular signals responses, and cellular relations with the extracellular bone matrix and different biomaterials.29,30,39,40 More issues have yet to be clarified as to the role of physical and chemical biomaterial properties in osteointegration processes and the main characteristics able to influence the bone healing and growth.31,32 In the present research, the different characteristics and behavior of 2 different biomaterials, Bovine Bone Grafts (Bio-Oss) and Hydroxyapatite (HA) ceramic scaffolds in presence of human OCs during the initial phases of bone development were evaluated.
MATERIALS
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Human Dental Papilla Stem Cells Expansion Culture
Four healthy patients (2 boys and 2 girls), average age of 11 years, were recruited at the Oral Science Department of Chieti University, for the third molar germ extraction for orthodontic treatment. In accordance with the Ethics Committee of the University of Chieti, all patients (or their parents in case of minors) expressed informed written consent before surgery. Dental follicles were delicately separated from the germs, immediately after tooth extraction, and were isolated. The samples were digested in a solution of a-Minimal Essential Medium (MEM) (Sigma, Milan, Italy) containing 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen, Milan, Italy), 500 mg/mL clarithromycin (Menarini, Florence, Italy), 3 mg/mL collagenase type 1 (Sigma), and 4 mg/mL dispase (Roche, Monza, Italy) for 1 hour at 37°C. The cells, after a separation with filtering
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through a 70-mm strainer (Falcon; Becton Dickinson, Franklin Lakes, NJ), were resuspended in growth medium (MEM, 100 mM 2-phospho-L-ascorbic acid, 2 mM L-glutamine [Sigma], 100 U/mL penicillin, 100 mg/mL streptomycin) supplemented with 20% fetal bovine serum (FBS; Invitrogen), and centrifuged for 10 minutes at 1200 rpm. The pellet, inserted in the same medium, was plated in 25-cm2 flasks and incubated at 37°C in a 5% CO2. The medium was changed twice a week. Cell proliferation analysis was performed by 3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) assay, using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Milan, Italy), according to the manufacturer’s instructions. The absorbance was measured at 490 nm, using a microtiter plate reader (Spectracount; Perkin Elmer Life, Waltham, MS). In addition, the number of viable cells was detected by the trypan blue exclusion test. At the indicated time, cells were trypsinized, added with trypan blue (0.15%) for 5 minutes, and then counted by a hemocytometer under optical microscope. FACS Analysis and Mesenchymal Stem Cell Sorting
Primary cultured cells were collected by trypsin/EDTA treatment, washed twice with PBS, and incubated with different monoclonal antibodies (CD14-FITC, CD15-FITC, CD29-PE, CD34-PE, CD45-PE, CD90-FITC, CD117-PE, CD146-PE, and CD166-PE from Becton Dickinson or STRO-1FITC from Santa Cruz Biotechnologies [Santa Cruz, CA]) at room temperature for 30 minutes in a dark room. The cells were then fixed and lysed with FACS lysing solution (Becton Dickinson), washed with PBS, and analyzed in a Becton Dickinson FACS flow cytometer using Cell Quest software. The calibration was carried out with 4 colors using Calibrite3 plus APC (Becton Dickinson) and analyzed by FACSComp software. In Vitro Osteogenic Differentiation and Alizarine Red Analysis
All the cell samples were cultured in osteogenic medium at a no less than 2 3 107/mL density. Adherent cells were trypsinized (0.025% trypsin/0.04%
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EDTA dissolved in phosphate-buffered saline [PBS], 10 minutes, 37°C) and seeded on culture onto 10-mm plates. After 3 days, replating cells were cultured in osteogenic medium adding to a-MEM culture medium, consisting of 15% FBS, 10 mM glycerol-2-phosphate disodium salt, 50 mg/mL 2-phospho-Lascorbic acid, and 10 nM dexamethasone (Sigma) as described by Owen and stay in these conditions under a controlled atmosphere (5% CO2, at 37°C) for 40 days. To assess the presence of mineralized depositions in the extracellular matrix, the differentiated cells culture were stained at days 5, 10, and 40 with Alizarin Red S solution (SigmaAldrich, Milan, Italy), according to the method described by Gregory et al. Runx2 Gene
The Runx2 gene has been identified as a member of the RUNX family of transcriptionfactors andencodesanuclear protein with the Runt DNA-binding domain. This protein is essential for osteoblastic differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression. The protein can bind DNA both as a monomer or, with more affinity, as a subunit of a heterodimeric complex. Mutations in this gene have been associated with the bone development disorder cleidocranial dysplasia (CCD). Transcript variants that encode different protein isoforms result from the use of alternate promoters as well as alternate splicing provided by RefSeq (UniProtKB/SwissProt: RUNX2-HUMAN, Q13950 Function: Transcription factor involved in the osteoblastic differentiation, essential for the maturation of osteoblast cells and skeletal morphogenesis). RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from adherent cells using TRIzol reagent (Life Technologies, Carlsbad, CA), according to the manufacturer’s recommendations. The resulting RNA pellet was air-dried and redissolved in 30 mL diethyl-pyrocarbonate–treated water. The quantity and purity of RNA were estimated spectrophotometrically by
IMPLANT DENTISTRY / VOLUME 22, NUMBER 6 2013 absorbance at 260 nm, and 5 mg were run on formaldehyde denaturing gel to confirm the integrity of the RNA. To remove any genomic DNA contaminants, RNA samples (10 mg) were treated with 1 U DNase-I RNase–free (RocheSwiss). First strand cDNA was synthesized from 1.5 mg of total RNA using the reverse transcription-polymerase chain reaction (RT-PCR) system RETROscript (Applied Biosystems, Milan, Italy) with random hexamers. The resultant cDNA (2 mL) was amplified in a 100-mL reaction volume containing PCR reaction buffer, 1.5 mM MgCl2, 0.2 mM each deoxy-dNTP, 1 mM oligonucleotide primers (MWG Biotech, Ebersberg, Germany), and 2.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The following human primers were used: alkaline phosphatase (ALP; Accession Number BC066116.1) forward primer: 50 TCACTCTCCGAGATGGTGGT-30 , reverse primer: 50 -ACTGCGCCTGGTAGTTGTTGT-30 ; MEPE (Accession Number NM_020203.1) forward primer: 50 -GGCAGCGGTTATACAGATCTT30 , reverse primer: 50 -ATGGGCAGGCCCTGACTTTT-30 ; osteocalcin (OCN; Accession Number NM_199173) forward primer: 50 -ACACCATGAGAGCCCTCACA-30 , reverse primer: 50 -ATGATGGGGACCCCACAT-30 ; GAPDH (Accession Number NM_002046) forward primer: 50 ACCACAGTCCATGCCATCAC-30 , reverse primer: 50 TCCACCACCCTGTTGCTGTA-30 . Conditions applied for PCR amplification were 94°C for 5 minutes, 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C (ALP and MEPE) or 58°C (OCN and GAPDH) for 1 minute, and elongation at 72°C for 1 minute. Reaction was also performed without the reverse transcriptase step as a control for genomic contamination. Amplification products were resolved by 1.5% agarose gel electrophoresis. The identity of the products was confirmed by cycle sequencing the amplified cDNA. The forward primer (50 -ATGCGTATTCCCGTAGATCCG AG-30 ) and reverse primer (50 -GCCG GGTGGTCGGCGATGATCT-30 ) of Runx2 were synthesized based on the hCbfa1/I (AML3) mRNA sequence (GenBank accession No. XM044800).
To identify the Runx2 (hCbfa1/II isoform), the forward primer (50 -ATGC TTCATTCGCCTCACAAAC-30 ) and reverse primer (50 -CCAAAAGAAGTTTTGCTGACATGG-30 ) were synthesized based on the 50 -untranslated region and the coding sequence of the hCbfa1/II mRNA, respectively. Amplifications were performed using a GeneAmp 9600 thermal cycler (Perkin-Elmer, Shelton, CT), with the temperature cycling being set as 94°C for 30 seconds. PCR products were analyzed by 1.5% agarose gel electrophoresis containing 0.01% ethidium bromide. Visualized PCR product bands were sliced from the gel, and radioactivity within the gel was detected using a Beckman LS6000 scintillation counter (Beckman Instruments, Fullerton, CA). RNA samples prepared from duplicate cultures were analyzed for each gene at each condition, and quantification of cDNA was normalized to GAPDH values. Northern Blot Hybridization
The total RNA (10 mg) was resolved on a 1% formaldehyde-agarose gel and transferred to a Hybond N+ nylon membrane (Amersham) by capillary blotting. The cDNA probe 50 -untranslated and coding sequence (0.3 kb) was prepared as described previously, and 50 ng of the cDNA was radiolabeled with [32P]dCTP to a specific activity of .109 cpm per microgram DNA using a random primer DNA labeling kit. Hybridization was carried out overnight at 42°C, and after stringent washing (twice for 10 minutes at room temperature in 33 standard SSC and 0.1% sodium dodecyl sulfate [SDS]; twice for 15 minutes at 43°C in 13 SSC and 0.1% SDS), membranes were subjected to autoradiography at −80°C. Band intensity was quantitatively analyzed on an imaging densitometer and normalized with control hybridization with GAPDH cDNA. Western Blotting Analysis
Cells were collected at 4°C in a lysis buffer (Tris buffer 50 mM, NaCl 150 mM, PMSF 1.0 mM; 1% Nonidet P40, 5 mg/mL leupeptin, 5 mg/mL aprotinin) at the indicated times, disrupted by sonication and centrifuged (14,000 rpm, 5 minutes, 4°C). Protein concentration was determined by BioRad protein assay
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(Bio-Rad Laboratories, Milan, Italy). Samples (50 mg), diluted in SDSbromophenol blue buffer, were boiled (5 minutes) and separated on 12.5% to 15% SDS–polyacrylamide gels. Proteins were transferred onto a polyvinylidene fluoride membrane, blocked with PBS/0.1% Tween20/5% nonfat milk (Bio-Rad Laboratories) for 2 hours at 4° C, incubated overnight at 4°C with specific primary antibodies (polyclonal rabbit anti-human ALP dilution 1:10,000, anti-human OCN dilution 1:200 [Abcam, Cambridge, United Kingdom], and anti-human MEPE 1 mg/m [R&D, Minneapolis, MN}]) for 1 hour at room temperature and then repeatedly washed and exposed to donkey anti-rabbit HPRconjugated secondary antibody for 1 hour at room temperature (GE Healthcare Life Sciences; final dilution, 1:5000). To determine the equal loading of samples per lane, the blots were stripped and reprobed with an anti–b-actin antibody (dilution 1:100, incubation for 1 hour at room temperature; Santa Cruz Biotechnologies). Immunocomplexes were visualized using the enhancing chemiluminescence detection system (GE Healthcare Life Sciences) and quantified by densitometric analysis (Molecular Analyst System; Bio-Rad Laboratories). Statistical Analysis
All values were expressed as mean 6 standard error of the mean (s.e.m.). Statistical analyses were performed using 1-way analysis of variance followed by Duncan post hoc test. Significant differences (P , 0.05, P , 0.01, and P , 0.001) versus values detected in cells at 3 days after seeding (before the addition of the osteogenic supplements) are marked with different asterisks (*, **, and ***, respectively). Optical and Electronic Microscopy
Light microscope (LM) and scanning electron microscope (SEM) analyses of differentiating cells were performed, respectively, at 30 and 40 days (after the cell confluence). For LM analysis, cells were stained with Toluidine blue and observed under an Axiolab microscope (Carl Zeiss, Oberkochen, Germany) connected to a digital camera (Fuji FinePixS2Pro; FUJIFILM Corporation, Tokyo, Japan). The images were
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The samples were gradually dehydrated in increasing concentrations of propylene oxide (from 50% to 100%, 10% steps), saturated with amyl acetate, carried through critical point drying according to standard procedure using liquid carbon dioxide and sputtered with gold-palladium coating. Biomaterials
Highly porous natural scaffold materials (Bio-Oss). Deproteinized sterilized bovine bone with 75% to 80% porosity was used and with a crystal size of approximately 10 nm in the form of cortical and cancellous particles (Bio-Oss; Geistlich Biomaterials, BadenBaden, Germany). This biomaterial exhibits the same highly porous microarchitecture as human bone mineral (similar macroscopic and microscopic structure to human spongiosa bone). The crystallization dimension comes from 400 to 1000 Å. The inner surface area of the material is approximately 100 m2/g, wide interconnecting pore system. Synthetic hydroxyapatite 3-dimensional ceramic scaffold (ENGIpore). Highly porous 3-dimensional synthetic hydroxyapatite ceramic scaffold with 70% to 90% porosity was used and with a scaffold crystal size of approximately 200 to 500 mm (ENGIpore; Fin-Ceramica Faenza, Faenza, Italy). This biomaterial exhibits similar highly porous microarchitecture as human bone macroscopic and microscopic structure. The interconnecting pore system is approximately range area of the 80 to 200 mm. Fig. 1. Results of the FACS analysis of HDPaC MSC population. A, Graphs report the MSCs population with positive mesenchymal markers of CD90-CD29-CD166-STRO-1. B, Positive and negative markers are summarized in the table.
Fig. 2. Alizarin Red results after 5 and 10 days.
stored in RAF format with 3032 3 2035 grid of pixels. The cells were then observed by scanning electron microscope (SEM) analysis with a SEM LEO 435 VP after being subjected to fixing procedure: the cells were treated with 3% glutaraldehyde in PBS 0.15 M (pH ¼ 7.4) for 30 minutes and then washed in PBS 0.15 M for 15 minutes and postfixed in 2% phosphate-buffered osmium with the addition of sucrose 0.15 M, at room temperature for 5 hours.
Proliferation of OCs Onto Biomaterials
After the differentiation phase, OCs were replated onto the biomaterials test discs previously described. The cells were divided into 3 groups, and about 3.7 3 104 cells were seeded onto each of the 3 different test discs and cultured in standard growth medium for a total of 20 days and were observed by SEM.
RESULTS Isolation and Characterization of Cultured Cells Derived From Human Dental Papilla
The immunophenotypic FACsorter cytofluorimetric analysis (FACS) was
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Fig. 3. PaMSCs Runx2 microarray analysis. It shows the complete complex gene network and the direct interaction of Runx2 gene with ALP gene during the initial phase of cells differentiation. The activation of RUNX2 function gene and the deactivation of MEPE gene with BMP2, TGFb3, and collagenous gene family were related with the activation of the OC differentiation processes.
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performed at week 1 to characterize the progeny of isolated cells from human dental papilla tissues (DPaMSC populations), using a wide number of markers antigens associated with different cell phenotypes. A main subpopulation of Human DPaMSCs could be detected. These cells did not react with hematopoietic markers, but they were found to be positive for cell surface antigens usually present in mesenchymal stem cells. Among the dental follicles obtained from different donors, the pattern of marker expression did not vary significantly. Data showed that the primary cultures of human dental papilla mesenchymal stem cells were negative for hematopoietic markers CD14 (monocyte/macrophage), CD15 (granulocyte/monocyte), CD45 (common leukocyte antigen), CD34 (hematopoietic stem/progenitor cells/endothelium), and CD117 (or c-kit, an early progenitor mesenchymal stem cell markers marker), whereas they were positive for markers present in mesenchymal cells, such as CD90 (Thy-1), CD166 (activated leukocyte cell adhesion molecule), STRO-1 (early mesenchymal progenitor marker), CD146 (a pericyte marker), and CD29 (integrin b-1) (Fig. 1). After the 7th DIV, the results showed great cell viability cultures and became confluent after only a week. Alizarin Red Staining
After confluence, the proliferating cells were evaluated with Alizarin red staining. The results confirmed the osteogenic differentiation and proliferation of the MSCs population and their increasing during time (Fig. 2). Runx2 of OCs on Bio-Oss/Engipore HA Scaffolds
Fig. 4. The MSCs RUNX2 Trypan Blue and MTS assay analysis after 3, 5, and 10 days with Bio-Oss and HA ENGIpore scaffolds. The RUNX2 data showed after 3 days in OCs on BioOss scaffolds higher levels expression and increased rapidly after 5 days with high levels of reaction at the 10th DIV days. In cells onto ENGIpore HA scaffolds, the RUNX2 showed a lower level expression after 5 days and the mRNA levels remained almost constant during osteoblastic differentiation and growth after 10 days. Such a behavior paralleled the pattern of cell proliferation and resulted faster in OCs on Bio-Oss scaffolds.
RUNX2 identified as a “master gene” controlling osteoblast differentiation with RUNX2 microarray analysis showing the complex gene network relationship. In data analysis, it was possible to observe the close RUNX2 gene interaction with ALP gene during the initial phase of cell differentiation. The activation of RUNX2 function gene and the deactivation of MEPE gene with BMP2, TGFb3, and collagenous gene family were related with the activation of the OCs differentiation processes (Fig. 3). Semiquantitative
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by the 5th DIV as for the mRNA content (Fig. 5, C). MEPE expression resulted more rapidly downregulated in differentiating Bio-Oss/OC samples than in HA/ OC (Fig. 5, D). MEPE time-dependent value paralleled that of the proliferation rate and resulted characteristic for each scaffold type, and the MEPE downregulation occurred at values statistically significant from the 5th DIV in Bio-Oss/OC, whereas in HA/OC, its downregulation could be observed from the 10th DIV. This particular behavior could be observed both by RT-PCR and Western Blot analysis. Light Microscope Analysis
Fig. 5. The DPaCs MSCs ALP, OCN, and MEPE Trypan Blue and MTS assay analysis after 3, 5, and 10 days with Bio-Oss and HA ENGIpore scaffolds. The proliferation rate, assayed by Trypan Blue and MTS, was faster in OCs on Bio-Oss than in ENGIpore HA scaffolds. Also, the ALP gene expression and the protein content resulted upregulated and faster in Bio-Oss/OC group (at the 5th DIV) than in HA/OC samples (at the 5th DIV). For both kinds of cells, ALP levels returned to similar control values at the 40th DIV. In cell culture, OCN expression and the proteic intracellular content gradually increased during the osteogenic stimulation period, and statistically significant differences between the 2 groups could be detected by the 5th DIV as for the mRNA content. MEPE expression resulted more rapidly downregulated in differentiating Bio-Oss/OC samples than in HA/OCs. MEPE time-dependent value paralleled that of the proliferation rate and resulted characteristic for each scaffold type, and the MEPE downregulation occurred at values statistically significant from the 5th DIV in Bio-Oss/OCs, whereas in HA/OCs, its downregulation could be observed from the 10th DIV.
RT-PCR analysis demonstrated that the expression of MEPE decreased and ALP and OCN mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype. The RUNX2 mRNA RT-PCR data showed initial expression in OCs on Bio-Oss scaffolds after 3 days and increased rapidly after 5 days with high levels of reaction at the 10th DIV days. In the same period, the RUNX2 demonstration in cells onto ENGIpore HA scaffolds showed initial expression only after 5 days and the mRNA levels remained almost constant during the osteoblastic differentiation and growth after 10 days (Fig. 4, A and B). Therefore, the OCs on Bio-Oss scaffolds behavior compared with ENGIpore samples showed a faster proliferation rate in the same time. Expression of Osteoblastic Markers Bio-Oss and HA/OC Samples
After 3 days, DPaMSCs was replating, in osteogenic medium, and the
proliferation rate and the expression/ intracellular content of selected osteoblastic markers such as ALP, OCN, Runt-related transcription factor 2 (Runx2), and matrix extracellular phosphoglycoprotein (MEPE) were observed. During the first week of differentiation in an osteogenic medium culture, the proliferation rate, assayed by Tripan Blue and MTS, was faster in OCs on BioOss than on ENGIpore HA scaffolds (Figs. 4, A and 5). Also, the gene expression and the protein content of ALP, measured respectively by RT-PCR and Western Blot, resulted in faster upregulated in the Bio-Oss/OC group (at the 5th DIV) than in HA/OC samples (at the 5th DIV). For both kinds of cells, ALP levels returned to similar control values at the 40th DIV (Fig. 5, B). On the contrary, OCN expression and the proteic intracellular content gradually increased during the osteogenic stimulation period, and statistically significant differences between the 2 groups could be detected
The cells, after osteogenic differentiation, were separately seeded onto BioOss and ENGIpore HA scaffolds, and after 30 days, it was possible to observe a large number of cells in contact in all the samples. These cells appeared interconnected with each other, arranged in a close reticular net. OCs with Bio-Oss samples were elongated with a centrally located nucleus morphology, spindle-shaped, and a homogeneous fibroblast-like appearance. On ENGIpore HA scaffold, the cells had clustered and it was possible to observe cuboid and star-shaped forms (Fig. 6, A and B). At higher magnification, the OCs showed different behavior on the BioOss and ENGIpore HA scaffolds, respectively. More close spindle-shaped cells and stellate-like cells were strongly attached and seeded onto the whole Bio-Oss scaffold surface in an intimate cell-biomaterial contact along the entire surface of the pore surface, and only small areas were not colonized by cells. In all the HA scaffold surfaces, it was possible to observe a small number of spindle-shaped cells and a greater number with globular cuboid and fibroblastlike cell morphology, and a small number of stellate cell lines. The cells showed a large number of long cytoplasmic processes and filopodia in the initial phase of contact with the biomaterial surface and also colonized the inner surface of the Bio-Oss scaffold through the interconnection pores (Fig. 6, C and D). SEM Analysis
After confluence, the OCs were cultured onto the Bio-Oss and HA
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Fig. 6. DPaCs MSCs LM analysis (toluidine blue stain) on the different scaffolds: A and B, BioOss; C and D, ENGIpore. Low (A and B) and high magnification (C and D). In all scaffolds, a great number of cells in contact, each other interconnected and arranged in a close reticular net, can be observed. In Bio-Oss, more close spindle-shaped and stellate-like cells, in an intimate cells-biomaterial contact along the entire surface, are observed. In HA ENGIpore scaffolds, a small number of spindle-shaped cell and a more large number of fibroblast, globular, and cuboid cells morphology, with small number of adjacent stellate cell lines is shown.
Fig. 7. DPaCs MSCs scanning electron microscopy analysis on the different scaffolds: A and B, Bio-Oss; C and D, ENGIpore. Low (A and B) and high magnificence (C and D). In all scaffolds, a large number of cells, ranging from 10 to 30 mm, with a multilevel network organization can be observed; in particular, a high interaction with the substrate (wide proliferation and extended network) on Bio-Oss and a smaller number of cells, of greater dimension with a typical fibroblast form, slightly interconnected, and flattened on the surface on HA.
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biomaterial scaffolds. The size of clusters increased gradually with culture time, and after 10 days, the SEM culture analysis showed a large number of the cells in intimate contact around the biomaterial granules in all samples. The cells showed typical spindle shape, long stellate morphology, ranged from 10 to 30 mm, and a multilevel network organization around biomaterial. At low magnification on Bio-Oss samples, it was possible to observe a close cell network on biomaterial scaffold with only small areas not colonized by cells. The cells showed a typical multilevel more closed network onto the biomaterial surface. In particular at low magnificence on Bio-Oss samples, it was possible to observe a high interaction with the substrate testified by a wide proliferation of cells and an extended network on biomaterial scaffold; the covering was very high and only small areas were not colonized by cells. At the same magnification, HA samples showed a smaller number of cells of greater dimension, between 20 and 60 mm, with typical fibroblast form, slightly interconnected, flattened on the surface, and a reduced interaction with the scaffold substrate tested with an uncovered large samples areas (Fig. 7, A and B). At higher magnification, in Bio-Oss samples, a great number of small and elongated OCs, average 10 mm, appeared in a wide network flattened onto the biomaterial surface, with a strong cell-to-cell adhesion and in intimate contact with the scaffold. It is remarkable that a cell multilayer organization with stellate and polygonal morphology and whole cytoskeleton cells interconnected with large number of filopodia between the cells and typical exocytose vesicles between 4 and 8 mm onto the cell surface. The cells showed an initial penetration into microrough surface of the Bio-Oss scaffold; between the granulae and on the cell surfaces, sporadic initial center of matrix deposition were detected. In the same conditions in ENGIpore HA scaffold, it is possible to observe a single flattened layer with a low number of cuboids and rhomboidal cells, with large designed morphology around 40 to 50 mm with small number of cytoplasmic filopodia and lamellipodia projections cell-to-cell
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in initial phase of attach onto the biomaterial surface. The absence of vesicles and matrix deposition center were detected (Fig. 7, C and D).
DISCUSSION Several in vitro and in vivo studies have demonstrated a close interaction of cultured cells with scaffold matrices.11–35 During tissue regeneration, an essential role was related with proper scaffold material designed to mimic all the characteristics of natural extracellular matrix.29 Bone tissue engineering is now developing strategies for repair and reconstruction loss tissue in the maxillofacial region. Despite, the widespread usage of autologous tissues for implantology and maxillofacial surgical procedures, the material research is still in progress.36 The FACS analysis results have detected a highly cell proliferation. Stem cell sorting showed the typical immunophenotypic characterization of mesenchymal stem cells. The human DPaMSCs did not reacte with hematopoietic markers CD14 (monocyte/macrophage), CD15 (granulocyte/monocyte), CD45 (common leukocyte antigen), CD34 (hematopoietic stem/progenitor cells/endothelium), and CD117 (or c-kit, an early progenitor MSC marker). The results were positive for cell surface antigens usually present in mesenchymal stem cells, such as CD90 (Thy-1), CD166 (activated leukocyte cell adhesion molecule), STRO-1 (early mesenchymal progenitor marker), CD146 (a pericyte marker), and CD29 (integrin b-1). The cytofluorimetric results, using specific antibodies to marker different human cellular types, confirmed the mesenchymal progenitor cells presence and excluded, in the primary undifferentiated culture, the hematopoietic progenitors cells.26,27 After 10 days, the Alizarine Red analysis showed a high stained, confirming the mesenchymal characteristics of the selected cell cluster, the complete osteogenic differentiation, and mineral deposition of extracellular matrix.3,37,38 At the same time, the SEM analysis remarked a different behavior of OCs with Bio-Oss samples compared with Engipore scaffold. Large areas of microroughness
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Bio-Oss surface was covered, and the cells were arranged in close-fitting multilayer structure typical arrangement of the initial differentiation phase, with large number of polygonal-shaped and cytoplasmic cell-to-cell and cell-to-biomaterial interconnection. In these samples, at higher magnification, a large amount of cell exocytosis vesicles and initial sporadic matrix deposition center on the cell surface were detected. This particular cell morphology suggests a wide intracytoplasmic protein biosynthesis and confirmed high metabolic cell activity. In the same condition onto the ENGIpore samples, it was evaluated large areas were uncovered by the OCs, a single cell layer interconnected with the scaffold and the absence of vesicles, and center of calcified deposits. It could be demonstrated that highly porous bovine hydroxyapatite provides a favorable scaffold for human OCs to attach, proliferate, and synthesize extracellular matrix. However, at 30 days, the LM analysis observed that the human OCs were in intimate interactions between all the granulate scaffolds. The cell growth in the presence of Bio-Oss and ENGIpore was regular in all the samples, in a conventional multilayer culture with a large number of OCs flattened and were close-fitting onto both scaffolds, confirmed the biocompatibility, the high osteoconductive biomaterials capability, and no cytotoxic effects of both material scaffolds. After 10 days, SEM analysis evaluated the higher OCs number onto the Bio-Oss between ENGIpore scaffolds. At higher magnification, more Bio-Oss scaffold areas were colonized from osteoblasts than ENGIpore. Moreover, the cells adhesion to the biomaterial showed a typical multilayer cell net with an intense polygonal-shaped cell attachment and scaffold colonization, stronger spindleshaped and a close contact between cells on Bio-Oss than Engipore samples. The highly porous BB hydroxyapatite scaffold supported cellular differentiation, and they generate extracellular matrix involved in initial phases of bone healing. However, at the same time and in in vitro experimental conditions, a different microstructure cell affinity was observed between 2 different biomaterial scaffolds, which was
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confirmed with morphological and biological data.39,40 Thus, also in DPaMCs, MEPE is the only marker that is downregulated when cell proliferation ends and osteogenic differentiation is triggered and it can be reasonably assumed as a more general index for osteoblast differentiation. Because Dentonin, a fragment of MEPE protein, may promote proliferation of MSCs in vitro, it is possible that this protein is downregulated as soon as the proliferation rate decreases. The experimental data showed higher duplication rate coupled to an earlier downregulation of MEPE, either before or after cell differentiation. Moreover, the data found the more rapid downregulation of MEPE in differentiating DPaMCs onto Bio-Oss samples and an earlier expression of the osteogenic markers, ALP, and OCN, compared with HADPsMC samples. The data are in agreement with findings showing MEPE downregulation along differentiation of human bone marrow or dental pulp derived. Human OCs differentiation is associated primarily with increases in ALP and OCN and Runx2 activity. The role of Runx2 gene in bone formation has been demonstrated that manifest a complete absence of osteogenesis in deficient mice4–9 and in the multiple skeletal abnormalities in patients with CCD associated with genetic mutations.7,8 The gene activity increased in a time-dependent manner with the development of the osteoblast phenotype. The biological results of ALP, OC, and RUNX2 markers showed an increased level of these proteins in both of samples during the differentiation process. Nevertheless in Bio-Oss samples, the OCs remarked a fast speed of the protein production rates when compared with Engipore OC samples in the same time. The early downregulation of MEPE protein related with a fast increasing rate of RUNX2, ALP, and OC protein production allowing to assess that the differentiation, proliferation, and growth rates on Bio-Oss samples were greater when compared with the other experimented biomaterials. Therefore, the biomaterials’ microstructure and characteristics might be a critical role during the initial stage of cellular differentiation and growth.
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CONCLUSION Several aspects must to be clarified concerning the timing of regeneration sites with different heterologous biomaterials scaffolds, the possibility of subjecting sites to a clinically adequate immediate loading during the rehabilitation procedure, and how much as both predictable in time the maintenance of the clinical results obtained. The microporosity may considerably increase the protein adsorption capability of HA as previously demonstrated. The porous materials must act as a scaffold for tissues regeneration, and they guide cell migration, allow the vascularization and support the stem cell proliferation, adhesion, and differentiation into trabecular bone. The physic-chemical characteristics of porous scaffold play a critical role during the initial phases of bone formation for the degree of interconnection of the pores and pore size modulate cellular ingrowths in the material evidencing osteoconductive properties. Moreover, the results indicate that both materials showed good biocompatibility characteristics and were clinically able to promote and support the bone regeneration process during orthopedic, maxillofacial, and oral surgical procedures. The different chemical and structural micro features seem to play a critical role in the osteointegration processes during the initial phase of bone healing as demonstrated by the morphological and biological markers results.
DISCLOSURE The authors claim to have no financial interest in any company or in any of the products mentioned in this article.
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29. Mangano C, Scarano A, Iezzi G, et al. Maxillary sinus augmentation using an engineered porous hydroxyapatite: A clinical, histological, and transmission electron microscopy study in man. J Oral Implantol. 2006;32:122–131. 30. Somanathan RV, Sim unek A. Evaluation of the success of beta-tricalciumphosphate and deproteinized bovine bone in maxillary sinus augmentation using histomorphometry: A review. Acta Medica (Hradec Kralove). 2006;49:87–89. 31. Boëck-Neto RJ, Gabrielli M, Lia R, et al. Histomorphometrical analysis of bone formed after maxillary sinus floor augmentation by grafting with a combination of autogenous bone and demineralized freeze-dried bone allograft or hydroxyapatite. J Periodontol. 2002;73: 26–70. 32. Aguirre Zorzano LA, Rodriguez Tojo MJ, Aguirre Urizar JM. Maxillary sinus lift with intraoral autologous bone and B-tricalcium phosphate: Histological
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A Comparison of Bovine Bone and Hydroxyapatite Scaffolds During Initial Bone Regeneration: An In Vitro Evaluation Filiberto Mastrangelo, MD, DDS, PhD,* Raimondo Quaresima, BEng,† Alfredo Grilli, MD,‡ Lucia Tettamanti, MD,§ Raffaele Vinci, MD,k Gilberto Sammartino, MD, DDS, PhD,¶ Stefano Tetè, MD, DDS, PhD,# and Enrico Gherlone, MD, DDS**
n modern implantology, worldwide attention has been placed on the macroscopic implant design and surface characteristics to obtain rapid and predictable results in osteointegration.1–3 However, the success of simple and complex rehabilitation depends on implant placement in adequate quality and quantity of bone.4,5 Moreover, it is not always possible to find the jawbone of the implant suitable for proper implant rehabilitation due to the loss of 1 or more teeth.6 The alveolar bone resorption and remodeling processes produced a rapid loss of the normal maxillary or jawbone architecture.7,8 Additional surgical technique to current clinical success has evolved including
I
*Young Researcher, Department of Oral Medical Science and Biotechnology, University of Chieti, Chieti, Italy. †Associate Professor of Biomaterial Engineering, Department of Civil Engineering, Architecture and Environment, University of L’Aquila, L’Aquila, Italy. ‡Ordinary Professor of Biology, Department of Oral Medical Science and Biotechnology, University of Chieti, Chieti, Italy; Leonardo da Vinci Telematic University, Torrevecchia Teatina (Chieti), Chieti, Italy. §Researcher of Pedodontics and Orthodontics, Department of Oral Science, Insubria University of Varese, Varese, Italy. kMaxillofacial Surgeon, Department of Oral Science, University Vita e Salute Milano, Milan, Italy. ¶Associate Professor of Maxillofacial Surgery, Department of Surgical Science, University Federico II Napoli, Naples, Italy. #Associate Professor of Oral Surgery, Department of Oral Medical Science and Biotechnology, University of Chieti, Chieti, Italy. **Ordinary Professor and Dean of Oral and Maxillofacial Surgery, Department of Oral Science, University Vita e Salute Milano, Milan, Italy.
Reprint requests and correspondence to: Mastrangelo Filiberto, MD, DDS, PhD, Department of Oral Science, University “G. d’Annunzio”, Chieti, Via dei Vestini 31, 66013 Chieti Scalo (CH), Italy, Phone: +39-3358390720, Fax: +39-0871-3554073, E-mail: fi[email protected] ISSN 1056-6163/13/02206-613 Implant Dentistry Volume 22 Number 6 Copyright © 2013 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0b013e3182a69858
Objectives: To evaluate the different behavior of 3-dimensional biomaterial scaffoldsdBovine Bone (BB; Bio-Oss) and Hydroxyapatite (HA; ENGIpore)dduring initial bone healing and development. Materials and Methods: Human dental papilla stem cells (hDPaSCs) were selected with FACsorter cytofluorimetric analysis, cultured with osteogenic medium, and analyzed with Alizarin red stained after differentiation. The obtained osteoblast-like cells (OCs) were cultured with BB and HA. alkaline phosphatase (ALP), OC, MEPE, and runt-related transcription factor 2 (RUNX2) expression markers were investigated performing Western blot and reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. After 40 days, samples were analyzed by light and electron microscopy.
Results: All the samples showed high in vitro biocompatibility and qualitative differences of OCs adhesion. RT-PCR and Western blot data exhibited similar marker rate, but ALP, OC, MEPE, and RUNX2expression, during initial healing and bone regeneration phase, was higher and faster in human dental papilla onto BB than in HA scaffolds. In biomaterials growth, RUNX2 seems to play an important role as a key regulator in human OCs from dental papilla bone development. Conclusion: Different surface BB scaffold characteristics seem to play a critical role in OCs differentiation showing different time of bone regeneration morphological characteristics as well as higher and faster levels of all observed markers. (Implant Dent 2013;22:613–622) Key Words: Bio-Oss, ENGIpore, bone, regeneration, osteogenesis, mesenchymal stem cells
the use of autologous or heterologous bone grafts to obtain bone regeneration and dental implant osteointegration.9,10 Several clinical studies have been performed to evaluate the use of different autologous or heterologous biomaterials scaffolds.11–13 Recent advances in clinical biotechnology research found interesting results of tissue regeneration, and several
reviews have critically analyzed the histological and clinical effects.12,13 Novel strategies for bone loss regeneration focus on tissue engineering, combine cell biology, and biomedical engineering, including stem cells and progenitor cells, bioactive scaffolds, and growth factors.14–16 Different biomaterials have been proposed and used for bone grafting: intraoral17,18 or
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extraoral19,20 autologous bone, allogenic grafts,21 xenografts,22,23 alloplastic grafts,24,25 or a combination of the same.26,27 The integration of these materials with the host tissue occurs by osteogenesis, osteoconduction, and osteoinduction.28 The main clinical advantage of bone regeneration with heterologous grafts was linked to the characteristics of the materials and the facilities to obtaining large quantities of materials.29 However, the different behavior between in vitro and in vivo tissue engineering development needs to be investigated as to progenitor cell biology, cell-to-cell interactions, molecular signals responses, and cellular relations with the extracellular bone matrix and different biomaterials.29,30,39,40 More issues have yet to be clarified as to the role of physical and chemical biomaterial properties in osteointegration processes and the main characteristics able to influence the bone healing and growth.31,32 In the present research, the different characteristics and behavior of 2 different biomaterials, Bovine Bone Grafts (Bio-Oss) and Hydroxyapatite (HA) ceramic scaffolds in presence of human OCs during the initial phases of bone development were evaluated.
MATERIALS
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METHODS
Human Dental Papilla Stem Cells Expansion Culture
Four healthy patients (2 boys and 2 girls), average age of 11 years, were recruited at the Oral Science Department of Chieti University, for the third molar germ extraction for orthodontic treatment. In accordance with the Ethics Committee of the University of Chieti, all patients (or their parents in case of minors) expressed informed written consent before surgery. Dental follicles were delicately separated from the germs, immediately after tooth extraction, and were isolated. The samples were digested in a solution of a-Minimal Essential Medium (MEM) (Sigma, Milan, Italy) containing 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen, Milan, Italy), 500 mg/mL clarithromycin (Menarini, Florence, Italy), 3 mg/mL collagenase type 1 (Sigma), and 4 mg/mL dispase (Roche, Monza, Italy) for 1 hour at 37°C. The cells, after a separation with filtering
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through a 70-mm strainer (Falcon; Becton Dickinson, Franklin Lakes, NJ), were resuspended in growth medium (MEM, 100 mM 2-phospho-L-ascorbic acid, 2 mM L-glutamine [Sigma], 100 U/mL penicillin, 100 mg/mL streptomycin) supplemented with 20% fetal bovine serum (FBS; Invitrogen), and centrifuged for 10 minutes at 1200 rpm. The pellet, inserted in the same medium, was plated in 25-cm2 flasks and incubated at 37°C in a 5% CO2. The medium was changed twice a week. Cell proliferation analysis was performed by 3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) assay, using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Milan, Italy), according to the manufacturer’s instructions. The absorbance was measured at 490 nm, using a microtiter plate reader (Spectracount; Perkin Elmer Life, Waltham, MS). In addition, the number of viable cells was detected by the trypan blue exclusion test. At the indicated time, cells were trypsinized, added with trypan blue (0.15%) for 5 minutes, and then counted by a hemocytometer under optical microscope. FACS Analysis and Mesenchymal Stem Cell Sorting
Primary cultured cells were collected by trypsin/EDTA treatment, washed twice with PBS, and incubated with different monoclonal antibodies (CD14-FITC, CD15-FITC, CD29-PE, CD34-PE, CD45-PE, CD90-FITC, CD117-PE, CD146-PE, and CD166-PE from Becton Dickinson or STRO-1FITC from Santa Cruz Biotechnologies [Santa Cruz, CA]) at room temperature for 30 minutes in a dark room. The cells were then fixed and lysed with FACS lysing solution (Becton Dickinson), washed with PBS, and analyzed in a Becton Dickinson FACS flow cytometer using Cell Quest software. The calibration was carried out with 4 colors using Calibrite3 plus APC (Becton Dickinson) and analyzed by FACSComp software. In Vitro Osteogenic Differentiation and Alizarine Red Analysis
All the cell samples were cultured in osteogenic medium at a no less than 2 3 107/mL density. Adherent cells were trypsinized (0.025% trypsin/0.04%
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EDTA dissolved in phosphate-buffered saline [PBS], 10 minutes, 37°C) and seeded on culture onto 10-mm plates. After 3 days, replating cells were cultured in osteogenic medium adding to a-MEM culture medium, consisting of 15% FBS, 10 mM glycerol-2-phosphate disodium salt, 50 mg/mL 2-phospho-Lascorbic acid, and 10 nM dexamethasone (Sigma) as described by Owen and stay in these conditions under a controlled atmosphere (5% CO2, at 37°C) for 40 days. To assess the presence of mineralized depositions in the extracellular matrix, the differentiated cells culture were stained at days 5, 10, and 40 with Alizarin Red S solution (SigmaAldrich, Milan, Italy), according to the method described by Gregory et al. Runx2 Gene
The Runx2 gene has been identified as a member of the RUNX family of transcriptionfactors andencodesanuclear protein with the Runt DNA-binding domain. This protein is essential for osteoblastic differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression. The protein can bind DNA both as a monomer or, with more affinity, as a subunit of a heterodimeric complex. Mutations in this gene have been associated with the bone development disorder cleidocranial dysplasia (CCD). Transcript variants that encode different protein isoforms result from the use of alternate promoters as well as alternate splicing provided by RefSeq (UniProtKB/SwissProt: RUNX2-HUMAN, Q13950 Function: Transcription factor involved in the osteoblastic differentiation, essential for the maturation of osteoblast cells and skeletal morphogenesis). RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from adherent cells using TRIzol reagent (Life Technologies, Carlsbad, CA), according to the manufacturer’s recommendations. The resulting RNA pellet was air-dried and redissolved in 30 mL diethyl-pyrocarbonate–treated water. The quantity and purity of RNA were estimated spectrophotometrically by
IMPLANT DENTISTRY / VOLUME 22, NUMBER 6 2013 absorbance at 260 nm, and 5 mg were run on formaldehyde denaturing gel to confirm the integrity of the RNA. To remove any genomic DNA contaminants, RNA samples (10 mg) were treated with 1 U DNase-I RNase–free (RocheSwiss). First strand cDNA was synthesized from 1.5 mg of total RNA using the reverse transcription-polymerase chain reaction (RT-PCR) system RETROscript (Applied Biosystems, Milan, Italy) with random hexamers. The resultant cDNA (2 mL) was amplified in a 100-mL reaction volume containing PCR reaction buffer, 1.5 mM MgCl2, 0.2 mM each deoxy-dNTP, 1 mM oligonucleotide primers (MWG Biotech, Ebersberg, Germany), and 2.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The following human primers were used: alkaline phosphatase (ALP; Accession Number BC066116.1) forward primer: 50 TCACTCTCCGAGATGGTGGT-30 , reverse primer: 50 -ACTGCGCCTGGTAGTTGTTGT-30 ; MEPE (Accession Number NM_020203.1) forward primer: 50 -GGCAGCGGTTATACAGATCTT30 , reverse primer: 50 -ATGGGCAGGCCCTGACTTTT-30 ; osteocalcin (OCN; Accession Number NM_199173) forward primer: 50 -ACACCATGAGAGCCCTCACA-30 , reverse primer: 50 -ATGATGGGGACCCCACAT-30 ; GAPDH (Accession Number NM_002046) forward primer: 50 ACCACAGTCCATGCCATCAC-30 , reverse primer: 50 TCCACCACCCTGTTGCTGTA-30 . Conditions applied for PCR amplification were 94°C for 5 minutes, 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C (ALP and MEPE) or 58°C (OCN and GAPDH) for 1 minute, and elongation at 72°C for 1 minute. Reaction was also performed without the reverse transcriptase step as a control for genomic contamination. Amplification products were resolved by 1.5% agarose gel electrophoresis. The identity of the products was confirmed by cycle sequencing the amplified cDNA. The forward primer (50 -ATGCGTATTCCCGTAGATCCG AG-30 ) and reverse primer (50 -GCCG GGTGGTCGGCGATGATCT-30 ) of Runx2 were synthesized based on the hCbfa1/I (AML3) mRNA sequence (GenBank accession No. XM044800).
To identify the Runx2 (hCbfa1/II isoform), the forward primer (50 -ATGC TTCATTCGCCTCACAAAC-30 ) and reverse primer (50 -CCAAAAGAAGTTTTGCTGACATGG-30 ) were synthesized based on the 50 -untranslated region and the coding sequence of the hCbfa1/II mRNA, respectively. Amplifications were performed using a GeneAmp 9600 thermal cycler (Perkin-Elmer, Shelton, CT), with the temperature cycling being set as 94°C for 30 seconds. PCR products were analyzed by 1.5% agarose gel electrophoresis containing 0.01% ethidium bromide. Visualized PCR product bands were sliced from the gel, and radioactivity within the gel was detected using a Beckman LS6000 scintillation counter (Beckman Instruments, Fullerton, CA). RNA samples prepared from duplicate cultures were analyzed for each gene at each condition, and quantification of cDNA was normalized to GAPDH values. Northern Blot Hybridization
The total RNA (10 mg) was resolved on a 1% formaldehyde-agarose gel and transferred to a Hybond N+ nylon membrane (Amersham) by capillary blotting. The cDNA probe 50 -untranslated and coding sequence (0.3 kb) was prepared as described previously, and 50 ng of the cDNA was radiolabeled with [32P]dCTP to a specific activity of .109 cpm per microgram DNA using a random primer DNA labeling kit. Hybridization was carried out overnight at 42°C, and after stringent washing (twice for 10 minutes at room temperature in 33 standard SSC and 0.1% sodium dodecyl sulfate [SDS]; twice for 15 minutes at 43°C in 13 SSC and 0.1% SDS), membranes were subjected to autoradiography at −80°C. Band intensity was quantitatively analyzed on an imaging densitometer and normalized with control hybridization with GAPDH cDNA. Western Blotting Analysis
Cells were collected at 4°C in a lysis buffer (Tris buffer 50 mM, NaCl 150 mM, PMSF 1.0 mM; 1% Nonidet P40, 5 mg/mL leupeptin, 5 mg/mL aprotinin) at the indicated times, disrupted by sonication and centrifuged (14,000 rpm, 5 minutes, 4°C). Protein concentration was determined by BioRad protein assay
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(Bio-Rad Laboratories, Milan, Italy). Samples (50 mg), diluted in SDSbromophenol blue buffer, were boiled (5 minutes) and separated on 12.5% to 15% SDS–polyacrylamide gels. Proteins were transferred onto a polyvinylidene fluoride membrane, blocked with PBS/0.1% Tween20/5% nonfat milk (Bio-Rad Laboratories) for 2 hours at 4° C, incubated overnight at 4°C with specific primary antibodies (polyclonal rabbit anti-human ALP dilution 1:10,000, anti-human OCN dilution 1:200 [Abcam, Cambridge, United Kingdom], and anti-human MEPE 1 mg/m [R&D, Minneapolis, MN}]) for 1 hour at room temperature and then repeatedly washed and exposed to donkey anti-rabbit HPRconjugated secondary antibody for 1 hour at room temperature (GE Healthcare Life Sciences; final dilution, 1:5000). To determine the equal loading of samples per lane, the blots were stripped and reprobed with an anti–b-actin antibody (dilution 1:100, incubation for 1 hour at room temperature; Santa Cruz Biotechnologies). Immunocomplexes were visualized using the enhancing chemiluminescence detection system (GE Healthcare Life Sciences) and quantified by densitometric analysis (Molecular Analyst System; Bio-Rad Laboratories). Statistical Analysis
All values were expressed as mean 6 standard error of the mean (s.e.m.). Statistical analyses were performed using 1-way analysis of variance followed by Duncan post hoc test. Significant differences (P , 0.05, P , 0.01, and P , 0.001) versus values detected in cells at 3 days after seeding (before the addition of the osteogenic supplements) are marked with different asterisks (*, **, and ***, respectively). Optical and Electronic Microscopy
Light microscope (LM) and scanning electron microscope (SEM) analyses of differentiating cells were performed, respectively, at 30 and 40 days (after the cell confluence). For LM analysis, cells were stained with Toluidine blue and observed under an Axiolab microscope (Carl Zeiss, Oberkochen, Germany) connected to a digital camera (Fuji FinePixS2Pro; FUJIFILM Corporation, Tokyo, Japan). The images were
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The samples were gradually dehydrated in increasing concentrations of propylene oxide (from 50% to 100%, 10% steps), saturated with amyl acetate, carried through critical point drying according to standard procedure using liquid carbon dioxide and sputtered with gold-palladium coating. Biomaterials
Highly porous natural scaffold materials (Bio-Oss). Deproteinized sterilized bovine bone with 75% to 80% porosity was used and with a crystal size of approximately 10 nm in the form of cortical and cancellous particles (Bio-Oss; Geistlich Biomaterials, BadenBaden, Germany). This biomaterial exhibits the same highly porous microarchitecture as human bone mineral (similar macroscopic and microscopic structure to human spongiosa bone). The crystallization dimension comes from 400 to 1000 Å. The inner surface area of the material is approximately 100 m2/g, wide interconnecting pore system. Synthetic hydroxyapatite 3-dimensional ceramic scaffold (ENGIpore). Highly porous 3-dimensional synthetic hydroxyapatite ceramic scaffold with 70% to 90% porosity was used and with a scaffold crystal size of approximately 200 to 500 mm (ENGIpore; Fin-Ceramica Faenza, Faenza, Italy). This biomaterial exhibits similar highly porous microarchitecture as human bone macroscopic and microscopic structure. The interconnecting pore system is approximately range area of the 80 to 200 mm. Fig. 1. Results of the FACS analysis of HDPaC MSC population. A, Graphs report the MSCs population with positive mesenchymal markers of CD90-CD29-CD166-STRO-1. B, Positive and negative markers are summarized in the table.
Fig. 2. Alizarin Red results after 5 and 10 days.
stored in RAF format with 3032 3 2035 grid of pixels. The cells were then observed by scanning electron microscope (SEM) analysis with a SEM LEO 435 VP after being subjected to fixing procedure: the cells were treated with 3% glutaraldehyde in PBS 0.15 M (pH ¼ 7.4) for 30 minutes and then washed in PBS 0.15 M for 15 minutes and postfixed in 2% phosphate-buffered osmium with the addition of sucrose 0.15 M, at room temperature for 5 hours.
Proliferation of OCs Onto Biomaterials
After the differentiation phase, OCs were replated onto the biomaterials test discs previously described. The cells were divided into 3 groups, and about 3.7 3 104 cells were seeded onto each of the 3 different test discs and cultured in standard growth medium for a total of 20 days and were observed by SEM.
RESULTS Isolation and Characterization of Cultured Cells Derived From Human Dental Papilla
The immunophenotypic FACsorter cytofluorimetric analysis (FACS) was
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Fig. 3. PaMSCs Runx2 microarray analysis. It shows the complete complex gene network and the direct interaction of Runx2 gene with ALP gene during the initial phase of cells differentiation. The activation of RUNX2 function gene and the deactivation of MEPE gene with BMP2, TGFb3, and collagenous gene family were related with the activation of the OC differentiation processes.
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performed at week 1 to characterize the progeny of isolated cells from human dental papilla tissues (DPaMSC populations), using a wide number of markers antigens associated with different cell phenotypes. A main subpopulation of Human DPaMSCs could be detected. These cells did not react with hematopoietic markers, but they were found to be positive for cell surface antigens usually present in mesenchymal stem cells. Among the dental follicles obtained from different donors, the pattern of marker expression did not vary significantly. Data showed that the primary cultures of human dental papilla mesenchymal stem cells were negative for hematopoietic markers CD14 (monocyte/macrophage), CD15 (granulocyte/monocyte), CD45 (common leukocyte antigen), CD34 (hematopoietic stem/progenitor cells/endothelium), and CD117 (or c-kit, an early progenitor mesenchymal stem cell markers marker), whereas they were positive for markers present in mesenchymal cells, such as CD90 (Thy-1), CD166 (activated leukocyte cell adhesion molecule), STRO-1 (early mesenchymal progenitor marker), CD146 (a pericyte marker), and CD29 (integrin b-1) (Fig. 1). After the 7th DIV, the results showed great cell viability cultures and became confluent after only a week. Alizarin Red Staining
After confluence, the proliferating cells were evaluated with Alizarin red staining. The results confirmed the osteogenic differentiation and proliferation of the MSCs population and their increasing during time (Fig. 2). Runx2 of OCs on Bio-Oss/Engipore HA Scaffolds
Fig. 4. The MSCs RUNX2 Trypan Blue and MTS assay analysis after 3, 5, and 10 days with Bio-Oss and HA ENGIpore scaffolds. The RUNX2 data showed after 3 days in OCs on BioOss scaffolds higher levels expression and increased rapidly after 5 days with high levels of reaction at the 10th DIV days. In cells onto ENGIpore HA scaffolds, the RUNX2 showed a lower level expression after 5 days and the mRNA levels remained almost constant during osteoblastic differentiation and growth after 10 days. Such a behavior paralleled the pattern of cell proliferation and resulted faster in OCs on Bio-Oss scaffolds.
RUNX2 identified as a “master gene” controlling osteoblast differentiation with RUNX2 microarray analysis showing the complex gene network relationship. In data analysis, it was possible to observe the close RUNX2 gene interaction with ALP gene during the initial phase of cell differentiation. The activation of RUNX2 function gene and the deactivation of MEPE gene with BMP2, TGFb3, and collagenous gene family were related with the activation of the OCs differentiation processes (Fig. 3). Semiquantitative
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by the 5th DIV as for the mRNA content (Fig. 5, C). MEPE expression resulted more rapidly downregulated in differentiating Bio-Oss/OC samples than in HA/ OC (Fig. 5, D). MEPE time-dependent value paralleled that of the proliferation rate and resulted characteristic for each scaffold type, and the MEPE downregulation occurred at values statistically significant from the 5th DIV in Bio-Oss/OC, whereas in HA/OC, its downregulation could be observed from the 10th DIV. This particular behavior could be observed both by RT-PCR and Western Blot analysis. Light Microscope Analysis
Fig. 5. The DPaCs MSCs ALP, OCN, and MEPE Trypan Blue and MTS assay analysis after 3, 5, and 10 days with Bio-Oss and HA ENGIpore scaffolds. The proliferation rate, assayed by Trypan Blue and MTS, was faster in OCs on Bio-Oss than in ENGIpore HA scaffolds. Also, the ALP gene expression and the protein content resulted upregulated and faster in Bio-Oss/OC group (at the 5th DIV) than in HA/OC samples (at the 5th DIV). For both kinds of cells, ALP levels returned to similar control values at the 40th DIV. In cell culture, OCN expression and the proteic intracellular content gradually increased during the osteogenic stimulation period, and statistically significant differences between the 2 groups could be detected by the 5th DIV as for the mRNA content. MEPE expression resulted more rapidly downregulated in differentiating Bio-Oss/OC samples than in HA/OCs. MEPE time-dependent value paralleled that of the proliferation rate and resulted characteristic for each scaffold type, and the MEPE downregulation occurred at values statistically significant from the 5th DIV in Bio-Oss/OCs, whereas in HA/OCs, its downregulation could be observed from the 10th DIV.
RT-PCR analysis demonstrated that the expression of MEPE decreased and ALP and OCN mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype. The RUNX2 mRNA RT-PCR data showed initial expression in OCs on Bio-Oss scaffolds after 3 days and increased rapidly after 5 days with high levels of reaction at the 10th DIV days. In the same period, the RUNX2 demonstration in cells onto ENGIpore HA scaffolds showed initial expression only after 5 days and the mRNA levels remained almost constant during the osteoblastic differentiation and growth after 10 days (Fig. 4, A and B). Therefore, the OCs on Bio-Oss scaffolds behavior compared with ENGIpore samples showed a faster proliferation rate in the same time. Expression of Osteoblastic Markers Bio-Oss and HA/OC Samples
After 3 days, DPaMSCs was replating, in osteogenic medium, and the
proliferation rate and the expression/ intracellular content of selected osteoblastic markers such as ALP, OCN, Runt-related transcription factor 2 (Runx2), and matrix extracellular phosphoglycoprotein (MEPE) were observed. During the first week of differentiation in an osteogenic medium culture, the proliferation rate, assayed by Tripan Blue and MTS, was faster in OCs on BioOss than on ENGIpore HA scaffolds (Figs. 4, A and 5). Also, the gene expression and the protein content of ALP, measured respectively by RT-PCR and Western Blot, resulted in faster upregulated in the Bio-Oss/OC group (at the 5th DIV) than in HA/OC samples (at the 5th DIV). For both kinds of cells, ALP levels returned to similar control values at the 40th DIV (Fig. 5, B). On the contrary, OCN expression and the proteic intracellular content gradually increased during the osteogenic stimulation period, and statistically significant differences between the 2 groups could be detected
The cells, after osteogenic differentiation, were separately seeded onto BioOss and ENGIpore HA scaffolds, and after 30 days, it was possible to observe a large number of cells in contact in all the samples. These cells appeared interconnected with each other, arranged in a close reticular net. OCs with Bio-Oss samples were elongated with a centrally located nucleus morphology, spindle-shaped, and a homogeneous fibroblast-like appearance. On ENGIpore HA scaffold, the cells had clustered and it was possible to observe cuboid and star-shaped forms (Fig. 6, A and B). At higher magnification, the OCs showed different behavior on the BioOss and ENGIpore HA scaffolds, respectively. More close spindle-shaped cells and stellate-like cells were strongly attached and seeded onto the whole Bio-Oss scaffold surface in an intimate cell-biomaterial contact along the entire surface of the pore surface, and only small areas were not colonized by cells. In all the HA scaffold surfaces, it was possible to observe a small number of spindle-shaped cells and a greater number with globular cuboid and fibroblastlike cell morphology, and a small number of stellate cell lines. The cells showed a large number of long cytoplasmic processes and filopodia in the initial phase of contact with the biomaterial surface and also colonized the inner surface of the Bio-Oss scaffold through the interconnection pores (Fig. 6, C and D). SEM Analysis
After confluence, the OCs were cultured onto the Bio-Oss and HA
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Fig. 6. DPaCs MSCs LM analysis (toluidine blue stain) on the different scaffolds: A and B, BioOss; C and D, ENGIpore. Low (A and B) and high magnification (C and D). In all scaffolds, a great number of cells in contact, each other interconnected and arranged in a close reticular net, can be observed. In Bio-Oss, more close spindle-shaped and stellate-like cells, in an intimate cells-biomaterial contact along the entire surface, are observed. In HA ENGIpore scaffolds, a small number of spindle-shaped cell and a more large number of fibroblast, globular, and cuboid cells morphology, with small number of adjacent stellate cell lines is shown.
Fig. 7. DPaCs MSCs scanning electron microscopy analysis on the different scaffolds: A and B, Bio-Oss; C and D, ENGIpore. Low (A and B) and high magnificence (C and D). In all scaffolds, a large number of cells, ranging from 10 to 30 mm, with a multilevel network organization can be observed; in particular, a high interaction with the substrate (wide proliferation and extended network) on Bio-Oss and a smaller number of cells, of greater dimension with a typical fibroblast form, slightly interconnected, and flattened on the surface on HA.
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biomaterial scaffolds. The size of clusters increased gradually with culture time, and after 10 days, the SEM culture analysis showed a large number of the cells in intimate contact around the biomaterial granules in all samples. The cells showed typical spindle shape, long stellate morphology, ranged from 10 to 30 mm, and a multilevel network organization around biomaterial. At low magnification on Bio-Oss samples, it was possible to observe a close cell network on biomaterial scaffold with only small areas not colonized by cells. The cells showed a typical multilevel more closed network onto the biomaterial surface. In particular at low magnificence on Bio-Oss samples, it was possible to observe a high interaction with the substrate testified by a wide proliferation of cells and an extended network on biomaterial scaffold; the covering was very high and only small areas were not colonized by cells. At the same magnification, HA samples showed a smaller number of cells of greater dimension, between 20 and 60 mm, with typical fibroblast form, slightly interconnected, flattened on the surface, and a reduced interaction with the scaffold substrate tested with an uncovered large samples areas (Fig. 7, A and B). At higher magnification, in Bio-Oss samples, a great number of small and elongated OCs, average 10 mm, appeared in a wide network flattened onto the biomaterial surface, with a strong cell-to-cell adhesion and in intimate contact with the scaffold. It is remarkable that a cell multilayer organization with stellate and polygonal morphology and whole cytoskeleton cells interconnected with large number of filopodia between the cells and typical exocytose vesicles between 4 and 8 mm onto the cell surface. The cells showed an initial penetration into microrough surface of the Bio-Oss scaffold; between the granulae and on the cell surfaces, sporadic initial center of matrix deposition were detected. In the same conditions in ENGIpore HA scaffold, it is possible to observe a single flattened layer with a low number of cuboids and rhomboidal cells, with large designed morphology around 40 to 50 mm with small number of cytoplasmic filopodia and lamellipodia projections cell-to-cell
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in initial phase of attach onto the biomaterial surface. The absence of vesicles and matrix deposition center were detected (Fig. 7, C and D).
DISCUSSION Several in vitro and in vivo studies have demonstrated a close interaction of cultured cells with scaffold matrices.11–35 During tissue regeneration, an essential role was related with proper scaffold material designed to mimic all the characteristics of natural extracellular matrix.29 Bone tissue engineering is now developing strategies for repair and reconstruction loss tissue in the maxillofacial region. Despite, the widespread usage of autologous tissues for implantology and maxillofacial surgical procedures, the material research is still in progress.36 The FACS analysis results have detected a highly cell proliferation. Stem cell sorting showed the typical immunophenotypic characterization of mesenchymal stem cells. The human DPaMSCs did not reacte with hematopoietic markers CD14 (monocyte/macrophage), CD15 (granulocyte/monocyte), CD45 (common leukocyte antigen), CD34 (hematopoietic stem/progenitor cells/endothelium), and CD117 (or c-kit, an early progenitor MSC marker). The results were positive for cell surface antigens usually present in mesenchymal stem cells, such as CD90 (Thy-1), CD166 (activated leukocyte cell adhesion molecule), STRO-1 (early mesenchymal progenitor marker), CD146 (a pericyte marker), and CD29 (integrin b-1). The cytofluorimetric results, using specific antibodies to marker different human cellular types, confirmed the mesenchymal progenitor cells presence and excluded, in the primary undifferentiated culture, the hematopoietic progenitors cells.26,27 After 10 days, the Alizarine Red analysis showed a high stained, confirming the mesenchymal characteristics of the selected cell cluster, the complete osteogenic differentiation, and mineral deposition of extracellular matrix.3,37,38 At the same time, the SEM analysis remarked a different behavior of OCs with Bio-Oss samples compared with Engipore scaffold. Large areas of microroughness
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Bio-Oss surface was covered, and the cells were arranged in close-fitting multilayer structure typical arrangement of the initial differentiation phase, with large number of polygonal-shaped and cytoplasmic cell-to-cell and cell-to-biomaterial interconnection. In these samples, at higher magnification, a large amount of cell exocytosis vesicles and initial sporadic matrix deposition center on the cell surface were detected. This particular cell morphology suggests a wide intracytoplasmic protein biosynthesis and confirmed high metabolic cell activity. In the same condition onto the ENGIpore samples, it was evaluated large areas were uncovered by the OCs, a single cell layer interconnected with the scaffold and the absence of vesicles, and center of calcified deposits. It could be demonstrated that highly porous bovine hydroxyapatite provides a favorable scaffold for human OCs to attach, proliferate, and synthesize extracellular matrix. However, at 30 days, the LM analysis observed that the human OCs were in intimate interactions between all the granulate scaffolds. The cell growth in the presence of Bio-Oss and ENGIpore was regular in all the samples, in a conventional multilayer culture with a large number of OCs flattened and were close-fitting onto both scaffolds, confirmed the biocompatibility, the high osteoconductive biomaterials capability, and no cytotoxic effects of both material scaffolds. After 10 days, SEM analysis evaluated the higher OCs number onto the Bio-Oss between ENGIpore scaffolds. At higher magnification, more Bio-Oss scaffold areas were colonized from osteoblasts than ENGIpore. Moreover, the cells adhesion to the biomaterial showed a typical multilayer cell net with an intense polygonal-shaped cell attachment and scaffold colonization, stronger spindleshaped and a close contact between cells on Bio-Oss than Engipore samples. The highly porous BB hydroxyapatite scaffold supported cellular differentiation, and they generate extracellular matrix involved in initial phases of bone healing. However, at the same time and in in vitro experimental conditions, a different microstructure cell affinity was observed between 2 different biomaterial scaffolds, which was
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confirmed with morphological and biological data.39,40 Thus, also in DPaMCs, MEPE is the only marker that is downregulated when cell proliferation ends and osteogenic differentiation is triggered and it can be reasonably assumed as a more general index for osteoblast differentiation. Because Dentonin, a fragment of MEPE protein, may promote proliferation of MSCs in vitro, it is possible that this protein is downregulated as soon as the proliferation rate decreases. The experimental data showed higher duplication rate coupled to an earlier downregulation of MEPE, either before or after cell differentiation. Moreover, the data found the more rapid downregulation of MEPE in differentiating DPaMCs onto Bio-Oss samples and an earlier expression of the osteogenic markers, ALP, and OCN, compared with HADPsMC samples. The data are in agreement with findings showing MEPE downregulation along differentiation of human bone marrow or dental pulp derived. Human OCs differentiation is associated primarily with increases in ALP and OCN and Runx2 activity. The role of Runx2 gene in bone formation has been demonstrated that manifest a complete absence of osteogenesis in deficient mice4–9 and in the multiple skeletal abnormalities in patients with CCD associated with genetic mutations.7,8 The gene activity increased in a time-dependent manner with the development of the osteoblast phenotype. The biological results of ALP, OC, and RUNX2 markers showed an increased level of these proteins in both of samples during the differentiation process. Nevertheless in Bio-Oss samples, the OCs remarked a fast speed of the protein production rates when compared with Engipore OC samples in the same time. The early downregulation of MEPE protein related with a fast increasing rate of RUNX2, ALP, and OC protein production allowing to assess that the differentiation, proliferation, and growth rates on Bio-Oss samples were greater when compared with the other experimented biomaterials. Therefore, the biomaterials’ microstructure and characteristics might be a critical role during the initial stage of cellular differentiation and growth.
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CONCLUSION Several aspects must to be clarified concerning the timing of regeneration sites with different heterologous biomaterials scaffolds, the possibility of subjecting sites to a clinically adequate immediate loading during the rehabilitation procedure, and how much as both predictable in time the maintenance of the clinical results obtained. The microporosity may considerably increase the protein adsorption capability of HA as previously demonstrated. The porous materials must act as a scaffold for tissues regeneration, and they guide cell migration, allow the vascularization and support the stem cell proliferation, adhesion, and differentiation into trabecular bone. The physic-chemical characteristics of porous scaffold play a critical role during the initial phases of bone formation for the degree of interconnection of the pores and pore size modulate cellular ingrowths in the material evidencing osteoconductive properties. Moreover, the results indicate that both materials showed good biocompatibility characteristics and were clinically able to promote and support the bone regeneration process during orthopedic, maxillofacial, and oral surgical procedures. The different chemical and structural micro features seem to play a critical role in the osteointegration processes during the initial phase of bone healing as demonstrated by the morphological and biological markers results.
DISCLOSURE The authors claim to have no financial interest in any company or in any of the products mentioned in this article.
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