In vitro analysis of the influence of mineralized and EDTA-demineralized allogenous bone on the viability and differentiation of osteoblasts and dental pulp stem cells

Bruno Machado Bertassoli . Gerluza Aparecida Borges Silva . Juliano Douglas Albergaria .Erika Cristina Jorge

Abstract

Grafting based on both autogenous and allogenous human bone is widely used to replace areas of critical loss to induce bone regeneration. Allogenous bones have the advantage of unlimited availability from tissue banks. However, their integration into the remaining bone is limited because they lack osteoinduction and osteogenic properties. Here, we propose to induce the demineralization of the allografts to improve these properties by exposing the organic components. Allografts fragments were demineralized in 10% EDTA at pH 7.2 solution. The influence of the EDTA-DAB and MAB fragments was evaluated with respect to the adhesion, growth and differentiation of MC30T3-E1 osteoblasts, primary osteoblasts and dental pulp stem cells (DPSC). Histomorphological analyses showed that EDTA-demineralized fragments (EDTA-DAB) maintained a bone architecture and porosity similar to those of the mineralized (MAB) samples. BMP4, osteopontin, and collagen III were also preserved. All the cell types adhered, grew and colonized both the MAB and EDTA-DAB biomaterials after 7, 14 and 21 days. However, the osteoblastic cell lines showed higher viability indexes when they were cultivated on the EDTA-DAB fragments, while the MAB fragments induced higher DPSC viability. The improved osteoinductive potential of the EDTA-DAB bone was confirmed by alkaline phosphatase activity and calcium deposition analyses. This work provides guidance for the choice of the most appropriate allograft to be used in tissue bioengineering and for the transport of specific cell lineages to the surgical site.

Keywords Bone grafts Cell adhesion and growth Cell differentiation Mesenchymal stem cells

Introduction

Bone tissue shows some regenerative capacity after injury. However, critical-size lesions, which may result from surgery, trauma, infection, or congenital malformation, require grafting to induce the growth and differentiation of a new and functional tissue. Bone grafting is a therapeutic option by which bonedeficient areas are built up with the use of different materials, such as autografts, allografts, alloplasts, and xenografts (Saima et al. 2016). When possible, autogenous bone is the preferred alternative, as it retains cell viability and does not induce any immunological response in the patient. In addition, it contains all three components needed for tissue engineering, including the scaffold, cells, and signalling molecules as an additional benefit (Pandit and Pandit 2016). However, autograft harvesting has limitations, including an additional surgery, donor site morbidity, and limited availability (Huber et al. 2017). Allogenous bone fragments are used to overcome the difficulties associated with autogenous grafts. Allografts are available from cadaveric bones as decellularized, chemically processed and frozen samples. The use of these allogenous fragments obtained from tissue banks reduces operating times and postoperative morbidity and allows for reconstructing large defect areas using only local anaesthesia (Garbin Junior et al. 2017).
Graftconsolidationintohostbonetissueismediated by three biological phenomena, including osteogenesis, osteoconduction and osteoinduction. Osteogenesis istheabilityoflivingcells(osteoblasts)tomaintainthe production of an osteoid substance, which occurs only with autologous grafts. Osteoinduction is the induction of host pluripotential cell differentiation into osteoblasts to establish a new bone. Osteoconductivity is the process in which the transplanted bone acts as a guide to induce the growth of osteoblast bridges of the new bone tissue, derived from the host. Osteoconductive and osteoinductive bone grafts can be used as scaffolds for currently existing osteoblasts while also triggering the formation of new osteoblasts, promoting faster integration of the graft (Saima et al. 2016). Autologous grafts are osteoconductive and osteoinductive and contain osteogenic cells, while allogenous grafts are only osteoconductive (Fillingham and Jacobs 2016).
One of the alternatives that is proposed to improve the osteoinductive properties of allogenous grafts is the exposure of the organic components of the bone matrix. The organic phase of the bone matrix harbors a full cocktail of bone growth factors, proteins, and other bioactive materials that are necessary for osteoinduction and successful bone healing. These include transforming growth factor-beta (TGF-b), insulin-like growth factors 1 and 2 (IGF1-2), platelet-derived growth factor (PDGF), fibroblast growth factor (FGFs) and bone morphogenic proteins (BMPs) (Linkhart et al. 1996). The chemical demineralization process of the bone blocks or trabeculae exposes these factors that induce bone cell proliferation and differentiation.
Tissue bone banks are now making allogenous grafts available in demineralized form, which is usually obtained by demineralization in acid solutions, such as hydrochloric acid (Figueiredo et al. 2011; Saima et al. 2016), following the Marshall Urist protocol (1965). In this condition, the mineral content of the bone is degraded, but the desired factors and proteins can be partially removed from the organic matrix. The protocol used to promote the demineralization of the allogenous grafts must preserve the osteogenic molecules to allow for the addition of the osteoinductive properties to this graft (Fillingham and Jacobs 2016). The demineralization of bone fragments by ethylenediamine tetraacetic acid (EDTA) might be one of these alternatives. A 10% EDTA solution is neutral (pH 7.2) and has been successfully used in bone histological procedures, preserving the collagenous and non-collagenous components of the organic matrix. The EDTA mechanism of action involves the capture and complexation of metal ions (such as calcium), removing them from the tissue with minimal histological changes (Hu¨lsmann et al. 2003).
In addition to attempting to improve intrinsic properties, tissue engineering principles also suggest that the association of the biomaterial with cells and organic factors adds osteoinduction potential to the allogenous bone fragments. Tissue engineering, an area related to regenerative medicine, is defined as the application of principles and techniques for the construction and growth of living tissue using biomaterials, cells and growth factors, by themselves or in combination. Bone tissue engineering research has advanced in order to associate different cell types and bioactivemoleculeswithsuchbiomaterials,alsocalled ‘‘scaffolds’’, for improving the osteogenesis and osteoinduction properties of their surfaces. In fact, theuseofbiomaterialsassociatedwithosteogeniccells shows relevant results in bone grafting (Wang et al. 2010; Hoffman et al. 2013; Liu et al. 2013; Shekaran et al. 2014; Li et al. 2016). The main cell lines used in bone tissue engineering are the MC3T3-E1 immortalized preosteoblast cell line, primary osteoblasts (PO) obtained from rat neonate calvaria, and mesenchymal stem cells (MSCs). The latter are indicated as the first choice in thesestudies dueto advantages such as ahigh differentiation potential, immunosuppressive effects, and viability after expansion in culture (Oryan et al. 2017). Among these mesenchymal precursors, dental pulp stem cells (DPSC) have excelled as a clonogenic cellular population, are highly proliferative, are capable of self-renewal and can differentiate into adipogenic, chondrogenic and osteogenic cell lines (Gronthos et al. 2000, 2002). DPSC differentiate into osteoblasts and produce bone tissue both in vitro and in vivo (Laino et al. 2005, 2006; d’Aquino et al. 2007; Li et al. 2011; Liu et al. 2011; Yuan et al. 2017). An important advantage of DPSC compared to other mesenchymal stem cells is that they are easily isolated from patients after a simple surgical procedure of extraction of primary teeth, molars or incisors (Tatullo et al. 2015), which can be removed in any age group. Many studies show that DPSC maintain their immnunophenotypic properties and differentiation potential even after a month of cryopreservation of a whole tooth (Tatullo et al. 2015). Thus, extracted human teeth, which are usually discarded as biological waste, could become an interesting source of cells for regenerative bone therapies.
In this work, we evaluated the influence of allogenous bone fragments, in their mineralized and EDTAdemineralized forms, on the adhesion, growth and differentiation of osteoblastic and dental pulp stem cells lineages. The comprehension of precursor cell behaviour when cultivated on these scaffolds allows for the selection of the most suitable allogenous matrix to be used in tissue bioengineering and to transport specific cell lineages, osteoblasts or DPSC, to the surgical site.

Materials and methods

Cell lines

MCT3E1 immortalized osteoblasts (IO), primary osteoblasts (PO) and dental pulp stem cells (DPSC) were used in this work. All the animal procedures were conducted after approval by the Ethics Committee on Animal Use (CEUA-UFMG). The MC3T3E1 immortalized osteoblasts (IO, American Type Culture Collection—ATCC) were commercially purchased. The primary osteoblasts (PO) were obtained from 5-day-old Wistar rat calvarias according to the protocol of Yamamoto et al. (2002). Briefly, calvarias were harvested, and the loose soft tissue was removed. The calvarias were digested with 0.1% Collagenase I (Gibco) and 0.05% Trypsin–EDTA (Gibco) in alpha-MEM (Sigma Aldrich). The digestion procedure was repeated twice. After centrifugation at 10009g for 5 min, the dissociated cells were resuspended in growth medium [alpha-MEM (Sigma Aldrich) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycinAmphotericin B (Anti-anti, Gibco) and were filtered through a 40 lm cell strainer.
Dental pulp stem cells (DPSC) were isolated from adult Wistar rat lower incisors according to the protocol described in Bertassoli et al (2016). Briefly, the pulps were removed through direct access of the apical foramen of the tooth. After digestion with 1.5% Collagenase I in alpha-MEM (Sigma Aldrich), the dissociated cells were resuspended in growth medium and were filtered through a 40 lm cell strainer. The data obtained from the DPSC isolation and characterization were previously published by our group (Bertassoli et al. 2016).
All the cells were cultivated in alpha-MEM supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin-Amphotericin B (Anti-anti, Gibco) at 37 C and in 5% CO2 and were used in the third passage. To evaluate the influence of the biomaterials on mineral deposition in all the cell lines, the growth medium was replaced after two days of culture with differentiation medium [alpha-MEM (Sigma Aldrich), supplemented with 10% FBS (Gibco), 50 mM ascorbic acid (Sigma Aldrich), 10 mM b-glycerophosphate, and 1% penicillin–streptomycin-Amphotericin B (Anti-anti, Gibco)] to induce cell differentiation and final maturation.

Biomaterials

The fragments of allogenous bone used in this work were obtained from the UNIOSS Tissue Bank (Skeletal Muscle Tissue Bank, Universidade de Marı´lia, Sa˜o Paulo, Brazil). These fragments were used in both commercial (mineralized allogenous bone—MAB) and EDTA-demineralized (EDTA-demineralized allogenous bone—EDTA-DAB) forms. All the bone allografts were fragmented to 0.5 9 0.5 9 0.5 cm using a dental device.
The EDTA-DAB fragments were obtained by incubating the MAB fragments in 10% EDTA at pH 7.2 for 48 h at room temperature. An energy-dispersive X-ray (EDX) analysis was performed to confirm the complete demineralization of the fragments (Fig. 1a). The MAB fragments were used as controls. The EDX analysis was performed using an X-ray diffractometer (Rigaku D/MAX, Japan) at the ICEX/ UFMG. As expected, calcium ions were found in the MAB fragments (Fig. 1a), which were used as the control, while no calcium was detected after the demineralization process (Fig. 1b). Carbon (C) and oxygen (O) were also detected in both samples as natural components, and osmium (Os) and gold (Au) ions were detected as residues of the processing of the samples for scanning electron microscopy (SEM).
After demineralization, the bone fragments were sterilized using gamma rays (25 grays for 30 min) at the Laborato´rio de Irradiac¸a˜o Gama/Centro de Desenvolvimento da Energia Nuclear (LIG/CDTN/UFMG). Before cell seeding, the fragments were histomorphologically characterized using a stereomicroscope, light microscope, and SEM.

Histomorphological analysis of the allografts Pore dimensions

Images of both the MAB and EDTA-DAB fragments were obtained using the stereomicroscope EZ4D (Leica) and the LAS EZ 2.1.0 software, at 35x. The pore dimensions were characterized following studied by Krishmamurithy and co-workers (Krishnamurithy et al. 2015). Measurements of the height (A – A0) and width (B – B0) of the visible pores (Fig. 3), from all six surfaces of all the blocks, were obtained using the ImageJ software. The measurements were plotted as percentage data. as residues of the processing of the samples

Surface

The ultrastructural aspects of the surface of both biomaterials were characterized by SEM. For this, the MAB and EDTA-DAB were rinsed with PBS, fixed in 4% glutaraldehyde, rinsed in PBS, and dehydrated in graded series of ethanol baths. After drying, the samples were mounted on aluminium stubs, gold sputtered and analysed using a DSM 950 (Zeiss, Germany) operating at an acceleration voltage of 5 kV.

Analysis of the organic matrix after demineralization with EDTA

The preservation of the organic constituents of the bone matrix after the EDTA action was evaluated by histology and immunofluorescence to detect BMP4, osteopontin, and collagen III, which are all markers of osteogenesis and bone repair processes. Collagen I was indirectly detected by eosin staining based on its acidophilic character. The EDTA-DAB fragments were fixed in 10% neutral buffered formalin, dehydrated in alcohol, diaphanized in xylene and embedded in paraffin. Serial sections of 5 lm were stained with haematoxylin and eosin and were analysed using a light microscope (Olympus BX-50).
For immunofluorescence, the sections were deparaffinized (xylene) and hydrated. After blocking with 2% BSA in Tris–HCl pH 7.4 for 1 h, the sections were incubated with primary antibodies as follows: anti-Osteopontin (Abcam, ab8448), diluted 1:200 in PBS; anti-BMP4 (Santa Cruz Biotechnology, sc6896), diluted 1:200 in PBS; and anti-type III Collagen (Abcam, ab7778), diluted 1:200 in PBS. After an overnight incubation at 4 C, the sections were rinsed with PBS and incubated at room temperature for 1 h in the dark with an appropriate conjugated secondary antibody as follows: anti-rabbit 488 or anti-goat 555 (Alexa Fluor—Molecular Probes), diluted 1:700 in 2% BSA in PBS. Nuclei were stained with DAPI (Life Technologies). The sections were washed with PBS and mounted with 80% glycerol. Imaging was performed using an Olympus BX50 microscope (Olympus, Hamburg, Germany).

3D cell culture

Osteoblasts (IO and PO) and DPSC were seeded at 5 9 104 onto fragments of MAB or EDTA-DAB in 40 ll of growth medium in 24-well plates. After 30 min of incubation at 37 C and 5% CO2, growth medium was added up to 500 ll. The cells were incubated at 37 C and 5% CO2 during all the experiments. The medium was replaced every two days. The samples designated for the mineral deposition analysis had their medium replaced with differentiation medium after two days of the initial incubation in growth medium.
The influence of both biomaterials in all the cell types was evaluated for cell adhesion and morphology by SEM, cell viability by MTT, cell differentiation by alkaline phosphatase activity, and cell maturation by alizarin red to detect mineral deposition. All the experiments were performed in triplicate.

SEM: cell adhesion and morphology analysis

A scanning electron microscope (SEM) analysis was used to characterize IO, PO and DPSC attachment and morphology on fragments of MAB and EDTA-DAB. After 7, 14 and 21 days of culture, the samples were processed as described for the analysis of the biomaterials surface.

MTT: cell viability analysis

Cell viability was assayed by MTT (Life Technologies), following the manufacturer’s protocol. Briefly, after 7, 14 and 21 days of cell culture onto MAB and EDTA-DAB, the bone constructs were carefully transferred to a new empty plate and 0.05 mg/ml of the MTT solution [3-(4,5-dimethylthiazol-2-yl)-2,5difeniltetrazolium-bromide] was added per well. The fragments were incubated at 37 C in 5% CO2 for 2 h. The MTT solution was then replaced by 500 lL of acid-isopropanol per well and was incubated on a shaker for 5 min. One-hundred microlitre samples from each well were transferred, in triplicate, to a 96-well plate, and the optical density (OD) was measured at 595 nm using a microplate reader (ELX800, BioTek).

Alkaline phosphatase activity: cell differentiation analysis

Alkaline phosphatase activity was detected using the NBT/BCIP kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The analysis was performed on IO, PO and DPSC cultivated on MAB and EDTA-DAB for 7 and 14 days. The cells were first rinsed with PBS, followed by an incubation with 200 ll/well of an NBT/BCIP solution at 1:1:8 ratio (NBT/BCIP/PBS) for 2 h at 37 C in 5% CO2. After incubation, 210 ll/well of SDS/0.1 N HCl 10% was added, and the plate was incubated for 18 h. Then, the solution was transferred to a 96-well plate (100 ll/ well), and the absorbance was measured at 595 nm. Alizarin Red: mineral deposition potential analysis
The formation of calcium phosphate was analysed using the Alizarin Red S assay. IO, PO and DPSC cells were cultivated onto MAD and EDTA-DAB, in differentiation medium, for 14 and 21 days.
The cells were rinsed with PBS and were fixed in 70% ethanol for 1 h at 4 C. The fixed cells were stained with 2% Alizarin Red S (pH 4.2) for 15 min at room temperature. The cells were rinsed with deionized water. The incorporated alizarin was quantified as described by Lin and co-workers (Lin et al. 2009). Briefly, 500 ll/well of 10% cetylpyridinium chloride was added and was incubated for 30 min with stirring. The final solution was transferred to a 96-well plate (100 ll/well), and the absorbency was measured at 550 nm. All the plates were photographed using an inverted phase contrast microscope (Motic AE31, USA).

Statistical analysis

Three or more experiments were performed for each proposed test, always using samples in triplicate. The results were analysed using the Prism software statistical software (GraphPad, San Diego, CA). The data were presented as the mean standard deviation and were compared statistically using a one-way ANOVA and/or a two-way ANOVA and Bonferroni test, using a confidence level of 95% (P\0.05).

Results

Biomaterial histomorphological aspects

Macroscopically, MAB presented an opaque aspect, with visible pores ranging in sizes and shape, and with regular trabeculae (Fig. 2a). After demineralization, the EDTA-DAB showed a translucent appearance, with a more irregular aspect of the trabeculae, compared to the MAB (Fig. 2b). These characteristics of the MAB and EDTA-DAB were more evident in the SEM analysis (Fig. 2c, d). An evident increase in the trabeculae size was observed in the EDTA-DAB (Fig. 2d).
The MAB and EDTA-DAB pore dimensions were also evaluated in this work. For the MAB, it was possible to note that the pore sizes ranged from 100 to 1000 lm, and 55% of the pores were [500 lm (Fig. 3), while the EDTA-DAB presented pores ranging from 300 to 1300 lm, and 76% of the pores were[500 lm (Fig. 3).
The histological analysis of the MAB fragments revealed homogeneous trabeculae, with the presence of cells with pyknotic nuclei well stained within and in the outer portion of the trabeculae. Collagen I fibers were can be noted as the pinkish tone stained by eosin (Fig. 4a).

Preservation of the organic matrix in the EDTA-DAB fragments

Sections of the DAB were immunostained for the presence of type III collagen, osteopontin and BMP4, which play important roles during osteogenesis. Representative images of sections of the human bone fragments showed the positive expression of all markers, including type III collagen, osteopontin and BMP4 markers (Fig. 4b–d), suggesting that the compounds of the bone matrix were maintained after the demineralization and decellularization processes. DAPI staining confirmed the presence of residual DNA in the trabeculae.
Influence of the biomaterials on cell adhesion and morphology showed an oval morphology onto both surfaces (Figs. 5a–f, 6a–f), while the DPSC showed a more fusiform morphology (Figs. 5g–i, 6g–i). All the cell lines established a continuous monolayer on the biomaterial, with adjacent cells contacting each other (Fig. 5).

Cell viability of the 3D culture

The MTT assay was used to evaluate the effects of the MAB and DAB biomaterials on cell viability. Cell viability was assayed after 7, 14 and 21 days of cell culture in growth media. An increase in IO, PO and DPSC cell viability was observed over time with both the MAB and EDTADAB biomaterials (Fig. 7). For IO and PO, the cell viability indexes were found to be significantly higher (P\0.05) on the cells cultivated on the EDTA-DAB biomaterial, over all three of the time points, compared to the data on the MAB fragments (Fig. 7a, b). The DPSC, however, showed a significantly higher cell viability (P\0.05) after 14 and 21 days of culturing on the MAB fragments compared with the indexes obtained from the cells on the DAB fragments (Fig. 7c).

Cell differentiation analysis

The potential of the MAB and EDTA-DAB biomaterials to induce IO, PO and DPSC differentiation were evaluated using the alkaline phosphatase activity assay. The enzyme activity was assayed after 7 and 14 days of cell culture in growth medium. The alkaline phosphatase activity was higher in the IO, PO and DPSC cultivated on the EDTA-DAB, both after 7 and 14 days of culture, compared to the data observed in the cells on the MAB (Fig. 8a–c).

Mineral deposition analysis

The potential of the MAB and EDTA-DAB biomaterials to induce IO, PO and DPSC mineralization was evaluated after 14 and 21 days of cell culture in differentiation medium. Similar to the observed results for the alkaline phosphatase activity, the EDTA-DAB biomaterials induced a higher calcium content in the IO, PO and DPSC cultures during both time points (Fig. 9a–c). The results were even more expressive in DPSC after 21 days (Fig. 9c).

Discussion

Bone tissue bioengineering has emerged as a new alternative to replace or induce bone repair, proposing the construction of a bone intermediate from a threedimensional culture of osteogenic or mesenchymal stem cells cultivated on biomaterials for subsequent transplantation in vivo. A number of different natural and synthetic biomaterials are being tested to allow for the construction of this bone intermediate, including bioactive glasses, bioceramics, titanium and chitosan (Orciani et al. 2017).
In this work, we evaluated fragments of allogenous bone that were demineralized by the action of EDTA as a proposal to optimize the osteoinductive properties of this biomaterial. Several studies reveal the successful use of allogenous bone fragments as biomaterials in their mineralized form, because of their osteoconductive and mechanical properties (Mauney et al. 2005; Al Kayal et al. 2015; Tollemar et al. 2016). However, allografts may have their osteoinductive properties unmasked by the demineralization process. According to Lekishvili et al (2004), inductive properties improve when the degree of their demineralization increases. As minerals are removed, the bone matrix exposes insoluble collagen (mostly type I with some types IV and X) and non-collagenous proteins (Mulliken et al. 1984), such as BMPs (Zhang et al. 1997). BMP2, BMP4, and BMP7 are associated with bone and mesenchymal cell differentiation and with the osteoinductivity properties of the demineralized bone matrices (DBM) (Blum et al. 2004; Marks et al. 2007; Murray et al. 2007). It is believed that the in situ diffusion of BMPs from DBM grafts is an integral step in their osteoinductive behaviour (Han 2008). The collagen structure, in turn, provides the osteoconductive effect of the demineralized allografts. Bone graft material that is osteoconductive and osteoinductive will not only serve as a scaffold for currently existing osteoblasts but also trigger the formation of new osteoblasts, promoting a faster integration of the graft (Saima et al. 2016).
The demineralized bone matrices are mainly produced by the extraction of the mineral components. To achieve this, the samples are normally immersed in a variety of strong and/or weak acids (Prasad and Donoghue 2013; Gupta et al. 2014). HCl, the most frequently used acid, induces the hydroxyapatite of the bone to form monocalcium phosphate and calcium chloride (Dorozhkin 1997; Horneman et al. 2004). HCl, at concentrations of 0.5–0.6 N, is the preferred demineralization solution due to its ability to remove bone mineral efficiently while still leaving the graft osteoinductive (Jain et al. 2004). BMPs are also generally recognized as being stable to such treatment. However, prolonged demineralization in acid can deplete the BMP concentration, probably through diffusion from the matrix into the acid bath (Pietrzak et al. 2011). The use of acidic solutions interferes with the osteoinductive properties of allogenous bone, inducing a variability that might impact the success of the clinical results.
Here, we propose the use of 10% EDTA at pH 7.2 as a chelating agent to expose the organic matrix. EDTA is the best calcium removal agent that also minimizes the loss of collagen/gelatine components. The superior results obtained with neutral EDTA may be attributed to the mechanism of capturing metallic ions (like calcium), which bind to the chelating agent. This means that the calcium ions from the external layer of the apatite crystals will be replaced by ions from the deeper layers. In this way, the crystal size decreases gradually, while preserving the tissue components (Sanjai et al. 2012). Unlike acid solutions, 10% EDTA at pH 7.2 demineralizes the bone matrix without the risks of inducing tissue damage by immersion in acid medium. This characteristic of this method favours the standardization of the samples routinely obtained from different grafting materials, such as cortical or cancellous bone, and distinct configurations, including long bones, blocks or particles with different shape and sizes.
The loss of the bone graft mineral components also induces a natural reduction of its mechanical properties (Al Kayal et al. 2015; Fillingham and Jacobs 2016; Tollemar et al. 2016). However, this loss might not interfere in the three-dimensional architecture and porosity of the graft. The pore dimensions and interconnectivity are essential features for nutrient diffusion and the removal of metabolic waste from cell activity (Huri et al. 2014). The porous size and organization also provide a microenvironment that influences cell migration, proliferation and differentiation (Huri et al. 2014). These morphological features are important for in vitro tissue construction as well as for promoting in vivo bone repair, as it favours cell penetration throughout the whole structure of the grafted fragment, allowing for uniform bone formation and better infusion of oxygen and culture medium penetration in the in vitro procedures (Krishnamurithy et al. 2015). In the present work, the EDTA-demineralized bone fragments maintained a bone architecture that was similar to the mineralized samples, preserving the porosity, with minimal morpho-structural changes. According to Karageorgiou and Kaplan (Karageorgiou and Kaplan 2005), the minimum pore size should be 100 lm, due the need for cell migration and transport. However, pore sizes[300 lm are recommended due the improvement in bone neoformation (Karageorgiou and Kaplan 2005; Krishnamurithy et al. 2015). The native trabecular bone shows a pore size ranging from 200 to 1500 lm (Grayson et al. 2010). Our analysis revealed that the MAB presented pore dimensions ranging from 100 to 1000 lm, while the EDTA-DAB showed pores ranging in size from 300 to 1,300 lm. These results demonstrated that both biomaterials agree with the recommended porosity parameters, which are considered optimal for cell growth.
To evaluate the effective gain in the osteoinductive properties for the MAB and EDTA-DAB scaffolds, we compared the potential of both to induce cell adhesion, growth and differentiation of two cellular lineages, osteogenic and dental pulp mesenchymal stem cells.
Since these cells are the ones responsible for the initial healing and eventually for new bone formation (Li et al. 2020), it is important that the proposed allografts are able to support their growth.
All the cell types (IO, PO and DPSC) adhered to, grew on and colonized both MAB and EDTA-DAB biomaterials during all three time points. In addition, all the cell lines established a continuous monolayer on the biomaterial, with adjacent cells contacting each other. A close adaptation of the monolayer to the surface of the MAB and EDTA-DAB biomaterial was also observed. These results showed that the processing promoted in these bone fragments preserved the ideal microenvironment to induce cell adhesion and proliferation. Cell adhesion to the biomaterial is a clue of the success of the later stages of the transplantation and bone repair.
MAB and EDTA-DAB induced different effects on the viability of each cell type. Osteoblastic cell lines (IO and PO) showed higher viability indexes when cultivated onto EDTA-DAB compared to MAB. The DPSC viability, however, was higher when these cells were cultivated on the MAB biomaterial. It is expected that the preference of the osteogenic cell lines (both IO and PO) to the exposed organic matrix (DAB) is because they need to attach to a structure composed of collagens (mostly type I but also types IV and X), noncollagenous proteins and growth factors, to undergo terminal differentiation by producing the inorganic matrix. The preference of the dental pulp stem cells for the MAB, however, suggested that the inorganic components of the bone matrix are important to promote undifferentiated stem cell survival, adhesion and growth. In fact, several studies show that mesenchymal stem cells easily adhere and proliferate in scaffolds of hydroxyapatite or those incorporated with other forms of calcium phosphate (Kweon et al. 2014; Domingos et al. 2017; Zima et al. 2017).
The improved osteoinductive potential of the DAB was confirmed by the analysis of the differentiation markers in IO, PO and DPSC. The alkaline phosphatase activity was higher in all the cell types cultivated in basal medium on the EDTA-DAB for 7 and 14 days compared to the MAB. Calcium deposition was also improved in these cells when they were cultivated on EDTA-DAB in osteogenic medium compared to MAB. The composition of the organic matrix, composed of a structure of type I collagen incorporated with osteogenic and growth factors, is clearly inducing osteogenic differentiation, including in the dental pulp mesenchymal stem cells.
In conclusion, both MAB and EDTA-DAB promoted positive effects on osteogenic and mesenchymal stem cells. However, MAB was ideal for promoting mesenchymal stem cell survival and proliferation, while the exposure of the organic matrix was important to improve the osteogenic characteristics. This work provided important biological information that will assist us in the choice of the best threedimensional structure and the particular cells to induce bone repair for different defects in vivo.

References

Al Kayal T, Panetta D, Canciani B, Losi P, Tripodi M, Burchielli S et al (2015) Evaluation of the effect of a gamma irradiated DBM-pluronic F127 composite on bone regeneration in wistar rat. PLoS ONE 10(4):e0125110
Bertassoli BM, Costa ES, Sousa CA, Albergaria JDS, Maltos KLM, Goes AM et al (2016) Rat dental pulp stem cells: isolation and phenotypic characterization method aiming bone tissue bioengineering. Braz Arch Biol Technol 59:e16150613
Blum B, Moseley J, Miller L, Richelsoph K, Haggard W (2004) Measurement of bone morphogenetic proteins and other growth factors in demineralized bone matrix. Orthopedics 27(1):S161–S165 d’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A et al (2007) Human postnatal dental pulp cells codifferentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ 14:1162–1171
Domingos M, Gloria A, Coelho J, Bartolo P, Ciurana J (2017) Three-dimensional printed bone scaffolds: The role of nano/micro-hydroxyapatite particles on the adhesion and differentiation of human mesenchymal stem cells. Proc Inst Mech Eng H 23:555–564
Dorozhkin SV (1997) Surface reactions of apatite dissolution. J Colloid Interface Sci 191:489–497
Figueiredo M, Cunha S, Martins G, Freitas J, Judas F, Figueiredo H (2011) Influence of hydrochloric acid concentration on the demineralization of cortical bone. Chem Eng Res and Des 89:116–124
Fillingham Y, Jacobs J (2016) Bone grafts and their substitutes. Bone Joint J 98B(1 Suppl A):6–9
Garbin Junior EA, de Lima VN, Momesso GAC, Mello-Neto JM, E´rnica NM, Magro Filho O (2017) Potential of autogenous or fresh-frozen allogeneic bone block grafts for bone remodelling: a histological, histometrical and immunohistochemical analysis in rabbits. Br J Oral Maxillofac Surg 55:589–593
Grayson WL, Frohlich M, Yeager K, Bhumiratana S, Chan ME, Cannizzaro C et al (2010) Engineering anatomically shaped human bone grafts. Proc Natl Acad Sci USA 107:3299–3304
Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 97:13625–13630
Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A et al (2002) Stem cell properties of human dental pulp stem cells. J Dent Res 81:531–535
Gupta S, Jawanda MK, Manjunath SM, Bharti A (2014) Qualitative histological evaluation of hard and soft tissue components of human permanent teeth using various decalcifying agents—a comparative study. J Clin Diag Res 8:ZC69–ZC72
Han B, Yang Z, Nimni M (2008) Effect of gamma irradiation on osteoinduction associated with demineralized bone matrix. J Orthop Res 26:75–82
Hoffman MD, Xie C, Zhang X, Benoit DS (2013) The effect of mesenchymal stem cells delivered via hydrogel-based tissue engineered periosteum on bone allograft healing. Biomaterials 34:8887–8898
Horneman DA, Ottens M, Hoorneman M, Van der Wielen LAM (2004) Reaction and diffusion during demineralization of animal bone. J Am Inst Chem Eng 50:2682–2690
Huber E, Pobloth AM, Bormann N, Kolarczik N, Schmidt-Bleek K, Schell H et al (2017) Demineralized bone matrix as a carrier for bone morphogenetic protein-2: burst release combined with long-term binding and osteoinductive activity evaluated in vitro and in vivo. Tissue Eng Part A 23:1321–1330
Huri PY, Ozilgen BA, Hutton DL, Grayson WL (2014) Scaffold pore size modulates in vitro osteogenesis of human adipose-derived stem/stromal cells. Biomed Mater 9(4):045003
Hu¨lsmann M, Heckendorff M, Lennon A (2003) Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J 36:810–830
Jain A, Kumar S, Aggarwal AN, Jajodia N (2015) Augmentation of bone healing in delayed and atrophic nonunion of fractures of long bones by partially decalcified bone allograft (decal bone). Indian J Orthop 49:637–642
Karageorgiou V, Kaplan D (2005) Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26:5474–5491
Krishnamurithy G, Murali MR, Hamdi M, Abbas AA, Raghavendran HB, Kamarul T (2015) Proliferation and osteogenic differentiation of mesenchymal stromal cells in a novel porous hydroxyapatite scaffold. Regen Med 10:579–590
Kweon H, Lee SW, Hahn BD, Lee YC, Kim SG (2014) Hydroxyapatite and silk combination-coated dental implants result in MC3 superior bone formation in the peri-implant area compared with hydroxyapatite and collagen combination-coated implants. J Oral Maxillofac Surg 72:1928–1936
Laino G, d’Aquino R, Graziano A, Lanza V, Carinci F, Naro F et al (2005) A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue (LAB). J Bone Miner Res 20:1394–1402
Laino G, Carinci F, Graziano A, d’Aquino R, Lanza V, De Rosa A et al (2006) In vitro bone production using stem cells derived from human dental pulp. J Craniofac Surg 17:511–515
Lekishvili MV, Snetkov AI, Vasiliev MG, Il’ina VK, Tarasov NI, Gorbunova ED et al (2004) Experimental and clinical study of the demineralized bone allografts manufactured in the tissue bank of CITO. Cell Tissue Bank 5:231–238
Li D, Deng L, Xie X, Yang Z, Kang P (2016) Evaluation of the osteogenesis and angiogenesis effects of erythropoietin and the efficacy of deproteinized bovine bone/recombinant human erythropoietin scaffold on bone defect repair. J Mater Sci Mater Med 27:101
Li JH, Liu DY, Zhang FM, Wang F, Zhang WK, Zhang ZT(2011) Human dental pulp stem cell is a promising autologous seed cell for bone tissue engineering. Chin Med J 124:4022–4028
Li J, Xu T, Hou W, Liu F, Qing W, Huang L et al (2020) The response of host blood vessels to graded distribution of macro-pores size in the process of ectopic osteogenesis. Mater Sci Eng C 109:110641
Lin L, Shen Q, Wei X, Hou Y, Xue T, Fu X et al (2009) Comparison of osteogenic potentials of BMP4 transduced stem cells from autologous bone marrow and fat tissue in a rabbit model of calvarial defects. Calcified Tissue Inter 85:55–65
Linkhart TA, Mohan S, Baylink DJ (1996) Growth factors for bone growth and repair: IGF, TGF beta and BMP. Bone 19:1S–12S
Liu HC, Ling-Ling E, Wang DS, Su F, Wu X, Shi ZP et al (2011) Reconstruction of alveolar bone defects using bone morphogenetic protein 2 mediated rabbit dental pulp stem cells seeded on nano-hydroxyapatite/collagen/poly(L-lactide). Tissue Eng Part A 17:2417–2433
Liu T, Wu G, Wismeijer D, Gu Z, Liu Y (2013) Deproteinized bovine bone functionalized with the slow delivery of BMP2 for the repair of critical-sized bone defects in sheep. Bone 56:110–118
Marks T, Wingerter S, Franklin L, Woodall J Jr, Tucci M, Russell G et al (2007) Histological and radiographic comparison of allograft substitutes using a continuous delivery model in segmental defects. Biomed Sci Instrum 43:194–199
Mauney JR, Jaquie´ry C, Volloch V, Heberer M, Martin I, Kaplan DL (2005) In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering. Biomaterials 26:3173–3185
Mulliken JB, Kaban LB, Glowacki J (1984) Induced osteogenesis: the biological principle and clinical applications. J Surg Res 37:487–496
Murray SS, Brochmann EJ, Harker JO, King E, Lollis RJ, Khaliq SA (2007) A statistical model to allow the phasing out of the animal testing of demineralized bone matrix products. Altern Lab Anim 35:405–409
Orciani M, Fini M, Primio RD, Belmonte MM (2017) Biofabrication and bone tissue regeneration: cell source, approaches and challenges. Front Bioeng Biotechnol 5:17
Oryan A, Kamali A, Moshiri A, Eslaminejad MB (2017) Role of mesenchymal stem cells in bone regenerative medicine: what is the evidence? Cells Tissues Organs 204:59–83
Pandit N, Pandit IK (2016) Autogenous bone grafts in periodontal practice: a literature review. J Int Clin Dent Res Organ 8:27–33
Pietrzak WS, Ali SN, Chitturi D, Jacob M, Woodell-May JE (2011) BMP depletion occurs during prolonged acid demineralization of bone: characterization and implications for graft preparation. Cell Tissue Bank 12:81–88
Prasad P, Donoghue M (2013) A comparative study of various decalcification techniques. Indian J Dent Res 24:302–308
Saima S, Jan SM, Shah AF, Yousuf A, Batra M (2016) Bone grafts and bone substitutes in dentistry. J Oral Res Rev 8:36–38
Sanjai K, Kumarswamy J, Patil A, Papaiah L, Jayaram S, Krishnan L (2012) Evaluation and comparison of decalcification agents on the human teeth. J Oral Maxillofac Pathol 16:222–227
Shekaran A, Garcı´a JR, Clark AY, Kavanaugh TE, Lin AS, Guldberg RE et al (2014) Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle. Biomaterials 35:5453–5461
Tatullo M, Marrelli M, Shakesheff KM, White LJ (2015) Dental pulp stem cells: function, isolation and applications in regenerative medicine. J Tissue Eng Regen Med9:1205–1216
Tollemar V, Collier ZJ, Mohammed MK, Lee MJ, Ameer GA, Reid RR (2016) Stem cells, growth factors and scaffolds in craniofacial regenerative medicine. Genes Dis 3:56–71
Urist MR (1965) Bone: formation by autoinduction. Science 150:893–899
Wang L, Fan H, Zhang ZY, Lou AJ, Pei GX, Jiang S et al (2010) Osteogenesisand angiogenesis of tissue-engineered bone constructed by prevascularized b-tricalcium phosphate scaffold and mesenchymal stem cells. Biomaterials 31:9452–9461
Yamamoto N, Furuya K, Hanada K (2002) Progressive development of the osteoblast phenotype during differentiation of osteoprogenitor cells derived from fetal rat calvaria: model for in vitro bone formation. Biol Pharm Bull 25:509–515
Yuan J, Liu X, Chen Y, Zhao Y, Liu P, Zhao L et al (2017) Effect of SOX2 on osteogenic differentiation of dental pulpstem cells. Cell Mol Biol 63:41–44
Zhang M, Powers RM, Wolfinbarger L (1997) Effects on the demineralization process on the osteoinductivity of demineralized bone matrix. J Periodont 68:1085–1092
Zima A, Czechowska J, Siek D, Olkowski R, Noga M, Lewandowska-Szumieł M et al (2017) How calcite and modified hydroxyapatite influence physicochemical properties and cytocompatibility of alpha-TCP based bone cements. J Mater Sci Mater Med 28:117