Surface characteristics of three commercially available grafts and adhesion of stem cells to these grafts

Abstract

Background:

Reconstruction of bone defects is often performed using bone autografts. However, limitations associated with the use of autografts led to the use of bone substitute materials.

Objectives:

The purpose of this study was to compare the surface characteristics of three commercially available grafts namely allografts, xenografts and alloplasts.

Methods:

This in vitro study was conducted on beta-tricalcium phosphate (β-TCP) alloplast, a mixture of demineralized bone matrix and mineralized bone allograft (DBM&MBA) and natural bovine bone mineral (NBBM) xenograft. Presence of apatite groups on the surface of samples was assessed by X-ray diffraction (XRD) while the presence of functional groups was evaluated using Fourier transform infrared spectroscopy (FTIR). Also, dental pulp stem cells (DPSCs) were cultured on the surface of samples and their adhesion was evaluated under a scanning electron microscope (SEM).

Results:

The FTIR showed a relatively similar pattern for NBBM and TCP samples and a different pattern in DBM&MBA. The results of XRD analysis also showed similarities between NBBM and TCP with sharper peaks than the DBM&MBA sample. The SEM micrographs showed that at 24 hours, no cell was detectable on the surface of NBBM sample; whereas, elongated cells were noted on the surface of TCP and DBM&MBA samples.

Conclusions:

The patterns of β-TCP and NBBM samples in XRD and FTIR spectroscopy showed high resemblance but they had different behaviors with respect to cell adhesion.

Keywords

Bone graft cell adhesion FTIR stem cells XRD

1. Introduction

Unlike some other tissues, bone is capable of regeneration. Injuries and bone fractures in many cases heal without scar formation [1,2]. However, in pathological fractures or extensive bone defects, bone regeneration may fail. Insufficient blood supply, infection of bone or the surrounding tissue and systemic conditions can negatively affect bone regeneration and result in the formation of non-homogenous structures or delay the formation of an integrated structure [3–6]. Bone graft refers to an implanted material that induces and enhances bone regenerations alone or in combination with some other materials [7]. This occurs via osteogenesis, osteoinduction and osteoconduction in combination or alone [8].

Materials used for bone grafts are grouped into three large categories of autografts, allografts and xenografts[9]. Each of these grafts has advantages and disadvantages. Allografts and xenografts have osteoinductive and osteoconductive properties but they lack the osteogenic properties of autografts [9–11]. Autografts are the gold standard for regeneration of small bone defects and have strong osteogenic properties with regard to bone regeneration, modeling and remodeling [12]. The need for a second surgery for graft harvesting, the associated risk of infection and hematoma [13,14], pain and donor site morbidity along with the risk of traumatizing the major vessels or organs when harvesting the graft are some of the shortcomings associated with the use of autografts [15]. Allograft is a non-vital bone tissue harvested from one individual and implanted in another individual of the same species. These grafts with different genotypes are harvested from fresh corpse and are available in fresh, frozen, freeze-dried and demineralized matrix forms [15–17]. Allografts have advantages such as availability, no need for a second surgery for graft harvesting, shorter duration of anesthesia, shorter surgical time and less blood loss. Risks of immune response and contamination of graft material have raised concerns with regard to the use of these materials. To eliminate these risks and overcome the existing limitations, the preparation protocol for the allogeneic graft materials is complicated; this protocol decreases the risk of infection transmission to one in eight million. The medical record of the donor must be checked for infection, malignancies, neoplasms, diseases affecting bone metabolism, hepatitis B and C, sexually transmitted diseases, autoimmune conditions and diseases affecting bone quality [15–17]. Xenografts are harvested from species other than humans (mammals and corals). Bovine hydroxyapatite has higher acceptability and application than other xenografts. However, the risk of disease transmission from animals to humans such as bovine spongiform encephalopathy and immune system stimulation must not be overlooked. Risk of transmission of such diseases is magnified when organic particles remain in the biomaterial. Thus, the process of procurement of xenografts is different from that of other graft materials; deproteinizing the bone minimizes its antigenicity [16 ,18].

Selection of an ideal bone graft depends on several factors such as tissue biocompatibility, defect size, graft size, shape and volume, biomechanical properties, handling of graft material, cost, ethical issues, biological properties and complications [9]. Considering the advantages and disadvantages of each graft material, comparison of different properties of bone grafts is useful. This in vitro study sought to compare the surface characteristics of three commercially available grafts using X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The adhesion of dental pulp stem cells (DPSCs) to the surfaces of these grafts was assessed as well.

2. Material and methods

2.1. Graft powders

Three commercially available bone graft powders namely beta-tricalcium phosphate (β-TCP), demineralized bone matrix and mineralized bone allograft (DBM&MBA), and natural bovine bone mineral (NBBM) were compared in this study; their characteristics are shown in Table 1.

Table 1
Characteristics of the three bone graft powders in this study

Graft type Product name Composition Manufacturer

Alloplast Cerasorb M Beta-tricalcium phosphate (TCP) Curasan-AG, Kleinostheim, Germany

Allograft DBM&MBA Freeze-dried human bone allograft ITB, Tehran, Iran

Xenograft Cerabone-Granulate Natural bovine bone matrix (NBBM) AAP Biomaterials GmbH, Dieburg, Germany

Graft type	Product name	Composition	Manufacturer
Alloplast	Cerasorb M	Beta-tricalcium phosphate (TCP)	Curasan-AG, Kleinostheim, Germany
Allograft	DBM&MBA	Freeze-dried human bone allograft	ITB, Tehran, Iran
Xenograft	Cerabone-Granulate	Natural bovine bone matrix (NBBM)	AAP Biomaterials GmbH, Dieburg, Germany

2.2. FTIR assessment

To determine the functional groups on the surface of samples, FTIR was carried out in the wavelength range of 400–4000 cm⁻¹ (IS10, Nicolet, USA).

2.3. XRD assessment

To determine the presence of apatite groups on the surface, XRD was performed at 1.57 CuKα wavelength (Equinox 3000, INEL, France).

2.4. Assessment of the seeding of DPSCs on the powders under SEM

Human DPSCs were isolated as described previously [19]. The fourth passage cells were used in this study; 20 mg/mL concentration of each powder was placed at the bottom of a 48-well plate and $2 \times 10^{5}$ DPSCs in 500 μL of standard culture medium (Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS) and 1% antibiotic and antimycotic) were also added. All samples were incubated at 37°C and 5% CO₂; 24 hours after cell culture, attached cells were evaluated under a SEM. For this purpose, the culture medium was extracted and the powders were transferred to another plate. They were washed twice with phosphate buffered saline (PBS) and fixed in 2.5% glutaraldehyde solution for two hours followed by one hour of immersion in 1% osmium. Dehydration was performed using a graded ethanol series (30, 50, 70, 90, 95 and twice in 100%). The samples were placed under a hood overnight and dried. The samples were gold-coated and observed under a SEM (EM3200, KYKY, China).

3. Results

3.1. FTIR assessment

Figure 1 shows the results of FTIR assessment of NBBM, TCP, and DBM&MBA samples. The results showed relatively similar FTIR patterns in NBBM and TCP samples. In DBM&MBA sample, absorption peaks were noted at 560, 1030, 1640 and 3440 cm⁻¹ wavelengths. The two peaks observed at 560 and 1030 cm⁻¹ wavelengths belonged to phosphate functional groups (PO₄ ⁻³) on the surface. The peaks in the range of 1450–1640 cm⁻¹ wavelengths belonged to carbonate (CO₃) and C–N and N–H bonds (seen only in DBM&MBA sample). The peak in the wavelength range of 3400–3600 cm⁻¹, might be attributed to the presence of hydroxyapatite as well as the amide N–H stretch.

Fig. 1.

Fourier transform infrared spectroscopy (FTIR) plot of the three bone grafts: beta-tricalcium phosphate (β-TCP), demineralized bone matrix and mineralized bone allograft (DBM&MBA), and natural bovine bone mineral (NBBM).

Fig. 1.

(Continued.).

In NBBM sample, all the afore-mentioned peaks except for those in the wavelength range of 1500–1700 cm⁻¹, which belonged to carbonate (CO₃) groups and C–N and N–H bonds were clearly visible. Carbonate groups in the range of 2000–2200 cm⁻¹ were more distinct in NBBM sample and the hydroxyl group (or the amide N–H) peak was well detectable at 3550 cm⁻¹ wavelength in NBBM sample.

In TCP sample, two peaks at 560 and 1030 cm⁻¹ wavelengths were clearly seen, which belonged to phosphate (PO₄ ⁻³) functional groups on the surface. Another peak was also noted in this group, which belonged to phosphate groups. Peaks indicative of hydroxyl groups or the amide N-H at 3550 cm⁻¹ wavelength were not seen in this sample.

3.2. XRD assessment

To analyze the crystalline inorganic (mineral) components, all samples were analyzed using XRD. Figure 2 shows the results of XRD analysis; as seen, the XRD pattern of DBM&MBA sample was different from that of the other two samples. Relatively similar XRD patterns were noted for NBBM and TCP; the peaks for these two samples were longer and more distinct than those of DBM&MBA sample.

Fig. 2.

X-ray diffraction (XRD) diagrams of the three bone grafts: beta-tricalcium phosphate (β-TCP), demineralized bone matrix and mineralized bone allograft (DBM&MBA), and natural bovine bone mineral (NBBM).

Fig. 2.

(Continued.).

3.3. Assessment of the seeding of DPSCs on the powders under SEM

Figure 3 shows the SEM micrographs obtained after 24 hours of culture of DPSCs on the three grafts. As seen, a few elongated cells were noted on DBM&MBA and TCP grafts at 24 hours but no cell was noted on NBBM graft.

Fig. 3.

SEM micrographs at 24 hours following the culture of dental pulp stem cells on the three bone grafts: beta-tricalcium phosphate (β-TCP) (SEM magnification: 2.00 kx), demineralized bone matrix and mineralized bone allograft (DBM&MBA) (SEM magnification: 2.50 kx), and natural bovine bone mineral (NBBM) (SEM magnification: 5.00 kx).

4. Discussion

At present, bone grafts are used as scaffolds to enhance bone regeneration. Autografts, allografts, xenografts and alloplasts can be used for this purpose, each having advantages and shortcomings. Obviously, materials that can serve as an osteoconductive scaffold and at the same time provide osteoinductive stimulation are preferred for bone regeneration [9–11]. In this study, an allograft, a xenograft and an alloplast were compared.

Evidence shows that the surface characteristics of grafts play an important role in their biological properties and function under in vivo and in vitro conditions [20]. Thus, accurate analysis of these characteristics is essential to predict their behavior. In this study, we first attempted to determine the presence of functional groups on the surface by FTIR and then performed XRD to determine the presence of apatite groups on the surface of samples. Assessment of the seeding of DPSCs on the surface of grafts was done using SEM.

The FTIR is an accurate method to determine the structural groups for indirect assessment of materials. In this technique, vibration of covalent bonds is performed using variable frequencies to detect functional groups present on the surface. This test is fast and affordable and can provide useful information about the chemical formulation of materials based on the intensity of the peaks and their width and shape in the required range of wavelengths [21]. In FTIR analysis, several distinct areas for phosphate groups were detected in all samples in our study. Presence of several phosphate peaks was related to symmetric and asymmetric stretching and bending molecular vibrations. Greenspan et al. compared IngeniOs HA Synthetic Bone Particles (which is a synthetic hydroxyapatite alloplast) with BioOss^® (deproteinized bovine bone) and found slight differences in the structure of these two materials by FTIR analysis. Similar to our study, phosphate peaks were seen in both groups. Moreover, a distinct carbonate peak was seen in Bio-Oss^® sample, which was probably due to the main components remaining or the result of the manufacturing process. This peak was not seen in IngeniOs sample [22]. In our study, the carbonate peak was seen in DBM&MBA sample while it was absent in the remaining two samples. Presence of carbonate (CO₃) in DBM&MBA sample indicated the replacement of some phosphate (PO₄) groups in bone structure with carbonate. These substitutions can cause significant changes in the solubility and biological properties of the materials [23]. An interesting finding was absence of hydroxyl group (OH⁻) peak in TCP sample while this peak was found in NBBM sample in the wavelength range of 3400–3600 cm⁻¹[21]. Elkayar et al. [24] derived hydroxyapatite from bovine teeth and showed PO₄ ⁻³ and OH⁻ peaks in FTIR analysis; this pattern was very much similar to the NBBM pattern in our study.

The XRD is a method to study the crystalline structure of solid materials; however, it is possible to detect different crystalline structures for materials with similar chemical composition [25]. Presence of wider peaks in XRD indicates low crystallinity while longer and sharper peaks show high crystallinity [25–27]. In our study, wider peaks in XRD pattern of DBM&MBA sample indicated the continuity of other functional groups, which decreased the crystalline phase [28]. Thus, dull pattern in this group indicates the presence of organic components in this material. Sharp peaks indicate well-deposited hydroxyapatite and absence of organic components [29–31]. The XRD patterns were relatively similar in NBBM and TCP samples. The peaks in these two groups were long and sharp. Longer and sharper peaks in these two samples indicated a more regular crystalline structure and higher percentage of crystallinity of these two samples. It should be noted that higher percentage of crystallinity is associated with lower solubility in the body [22]. Based on the results of FTIR, which is more accurate than XRD, it can be concluded that peaks in NBBM and TCP samples belonged to two different crystals; these peaks in NBBM sample belonged to hydroxyapatite while in TCP, they indicated β-TCP.

Kim et al. [32] compared AutoBT (regular tooth) with Bio-Oss^®, MBCP™ (composed of hydroxyapatite and TCP) and allogeneic bone, which resembled NBBM, TCP and DBM&MBA in our study, respectively; XRD analysis showed very similar patterns for AutoBT and allogeneic bone. However, XRD of MBCP™ sample showed sharper and more distinct peaks indicative of the higher crystallinity of this material. The same patterns were noted in the diagrams of NBBM and TCP samples. Studies have shown that higher crystallinity decreases solubility and results in consequently poorer biodegradation in the human body, which is not favorable considering the need for gradual replacement of scaffold with the newly formed bone in the human body [33].

In a study by Greenspan et al. [22] peaks related to the crystalline structure of synthetic hydroxyapatite (IngeniOs) were sharper than those of deproteinized bone, which indicates more regular crystalline structure and higher percentage of crystallinity of synthetic hydroxyapatite. This was also noted in our study in NBBM and TCP groups. In a study by Elkayar et al. [24], bovine tooth was deproteinized and calcination was performed at high temperature to derive hydroxyapatite from bovine tooth; the XRD pattern of the obtained sample was relatively similar to that of NBBM in our study.

The first step in cell-biomaterial interaction is cell adhesion to the surface of biomaterials, which can significantly affect the subsequent proliferation and differentiation steps [34]. Based on the results of SEM at one day after cell culture on powders, elongated cells with pseudopods were seen on the surface of DBM&MBA and TCP samples; but NBBM sample, which was a deproteinized xenograft had no cells. Pappalardo et al. compared demineralized and deproteinized bone in terms of adhesion of osteosarcoma cells. Demineralized bone showed superior adhesion of these cells. They attributed this finding to the rougher surface and granular structure of deproteinized compared to demineralized bone [34]. In our previous study, the attachment of DPSCs to NBBM was almost the same as that for freeze-dried bone allograft (under SEM); however, the proliferation of these cells was the lowest on NBBM (MTT assay) [35]. Seebach et al. compared six bone-graft substitutes with regard to cell seeding efficiency, on days 2, 6 and 10, and found the lowest number of cells on Cerabone (NBBM) during the whole observation period. Their quantitative data were confirmed by SEM [36].

In the study by Miron et al. NBBM demonstrated no potential for cell recruitment. Also, Handschel et al. reported very few cells on deproteinized bovine bone and their results were confirmed by SEM [37].

Naujoks et al. evaluated the biocompatibility of different biomaterials on day 1 and day 7 after cell-application with fluorescence-based microplate assay and SEM. The SEM results showed no cells observable on NBBM; whereas, on all other biomaterials, one layer of cells was visible [38]. Other authors also detected a close correlation between the proliferation assay and SEM in compatibility testing [39].

Researchers claim that smooth surface and low porosity of NBBM could be the reason for poor cell attachment, metabolism and growth behavior of cells on this biomaterial [36]; however, crystalized HA with even less porosities and smoother surface showed bioadhesive properties although only a few cells were observed [40].

In our previous study comparing deproteinized, demineralized and regular dentin using MTT assay and SEM, one group had suitable surface for cell proliferation in long-term; however at three days, no cell was seen on SEM micrographs of this group [41].

It seems that, even NBBM could be bioadhesive in long term however in the period examined in our study we did not see any cells on the surface of NBBM. Therefor one of the limitations of this study is absence of quantitative assays over an extended period of time for better comparison of these materials.

5. Conclusions

Based on the current results, β-TCP (alloplast), and NBBM (xenograft) samples were similar in terms of X-ray diffraction and Fourier transform infrared spectroscopy patterns but showed different behaviors with respect to cell adhesion. DBM&MBA (allograft) showed cell adhesion in 24 h but functional groups was not very intense in XRD and FTIR spectroscopy patterns.

Conflict of interest

The authors report no conflicts of interest related to this study.

References

Egermann

Lill

Griesbeck

Evans

Robbins

Schneider

and Baltzer

A.W.

, Effect of BMP-2 gene transfer on bone healing in sheep, Gene Ther 13(17) (2006), 1290–1299. doi:10.1038/sj.gt.3302785.

Nandi

Roy

Mukherjee

Kundu

and Basu

, Orthopaedic applications of bone graft & graft substitutes: a review, Indian J Med Res. 132 (2010), 15–30.

Bansal

Bhagat

and Shukla

, Bovine cancellous xenograft in the treatment of tibial plateau fractures in elderly patients, Int Orthop. 33(3) (2009), 779–784. doi:10.1007/s00264-008-0526-y.

Bigham

Dehghani

Shafiei

and Nezhad

S.T.

, Xenogenic demineralized bone matrix and fresh autogenous cortical bone effects on experimental bone healing: radiological, histopathological and biomechanical evaluation, J Orthop Traumatol. 9(2) (2008), 73–80. doi:10.1007/s10195-008-0006-6.

Hegde

Shetty

Wasnik

Ahammed

and Shetty

, Use of bone graft substitute in the treatment for distal radius fractures in elderly, Eur J Orthop Surg Traumatol. 23(6) (2013), 651–656. doi:10.1007/s00590-012-1057-1.

Scaglione

Fabbri

Dell’Omo

Gambini

and Guido

, Long bone nonunions treated with autologous concentrated bone marrow-derived cells combined with dried bone allograft, Musculoskelet Surg 98(2) (2014), 101–106. doi:10.1007/s12306-013-0271-2.

Tabatabaei

Motamedian

Gholipour

Khosraviani

and Khojasteh

, Craniomaxillofacial bone engineering by scaffolds loaded with stem cells: A systematic review, J Dent Sch. 30(2) (2012), 115–131.

Albrektsson

and Johansson

, Osteoinduction, osteoconduction and osseointegration, Eur Spine J. 10(Suppl 2) (2001), S96–S101. doi:10.1007/s005860100282.

Dimitriou

Jones

McGonagle

and Giannoudis

P.V.

, Bone regeneration: current concepts and future directions, BMC Med. 9 (2011), 66. doi:10.1186/1741-7015-9-66.

10.

Brydone

Meek

and Maclaine

, Bone grafting, orthopaedic biomaterials, and the clinical need for bone engineering, Proc Inst Mech Eng H. 224(12) (2010), 1329–1343. doi:10.1243/09544119JEIM770.

11.

Keskin

Gündoğdu

and Atac

A.C.

, Experimental comparison of bovine-derived xenograft, xenograft-autologous bone marrow and autogenous bone graft for the treatment of bony defects in the rabbit ulna, Med Princ Pract. 16(4) (2007), 299–305. doi:10.1159/000102153.

12.

Athanasiou

V.T.

Papachristou

D.J.

Panagopoulos

Saridis

Scopa

C.D.

and Megas

, Histological comparison of autograft, allograft-DBM, xenograft, and synthetic grafts in a trabecular bone defect: an experimental study in rabbits, Med Sci Monit. 16(1) (2010), BR24–BR31.

13.

Arrington

E.D.

Smith

W.J.

Chambers

H.G.

Bucknell

A.L.

and Davino

N.A.

, Complications of iliac crest bone graft harvesting, Clin Orthop Relat Res 329 (1996), 300–309. doi:10.1097/00003086-199608000-00037.

14.

Banwart

J.C.

Asher

M.A.

and Hassanein

R.S.

, Iliac crest bone graft harvest donor site morbidity: a statistical evaluation, Spine (Phila Pa 1976) 20(9) (1995), 1055–1060. doi:10.1097/00007632-199505000-00012.

15.

Ehrler

D.M.

and Vaccaro

A.R.

, The use of allograft bone in lumbar spine surgery, Clin Orthop Relat Res 371 (2000), 38–45. doi:10.1097/00003086-200002000-00005.

16.

Moshiri

and Oryan

, Role of tissue engineering in tendon reconstructive surgery and regenerative medicine: current concepts, approaches and concerns, Hard Tissue. 1(2) (2012), 11. doi:10.13172/2050-2303-1-2-291.

17.

Yazar

, Onlay bone grafts in head and neck reconstruction, Semin Plast Surg 24(3) (2010), 255–261. doi:10.1055/s-0030-1263067.

18.

Oryan

Alidadi

and Moshiri

, Current concerns regarding healing of bone defects, Hard Tissue 2(2) (2013), 13. doi:10.13172/2050-2303-2-2-374.

19.

Tabatabaei

F.S.

Jazayeri

Ghahari

and Haghighipour

, Effects of equiaxial strain on the differentiation of dental pulp stem cells without using biochemical reagents, Mol Cell Biomech. 11(3) (2014), 209–220.

20.

Suchanek

and Yoshimura

, Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants, J Mater Res. 13(1) (1998), 94–117. doi:10.1557/JMR.1998.0015.

21.

Berzina-Cimdina

and Borodajenko

, Research of calcium phosphates using Fourier transform infrared spectroscopy, in: INTECH, Open Access Publisher, 2012.

22.

Greenspan

D.C.

, Comparison of a synthetic and bovine derived hydroxyapatite bone graft substitute, Zimmer Dental Inc. (2013), 1–4.

23.

Shi

, Introduction to Biomaterials, World Scientific Pub Co Inc., 2006.

24.

Elkayar

Elshazly

and Assaad

, Properties of hydroxyapatite from bovine teeth, Bone Tissue Regen Insights 2(3) (2009), 31–36.

25.

Holzwarth

and Gibson

, The Scherrer equation versus the ‘Debye–Scherrer equation’, Nat Nanotechnol. 6(9) (2011), 534. doi:10.1038/nnano.2011.145.

26.

Balasundaram

Sato

and Webster

T.J.

, Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD, Biomaterials 27(14) (2006), 2798–2805. doi:10.1016/j.biomaterials.2005.12.008.

27.

Tadic

and Epple

, A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone, Biomaterials. 25(6) (2004), 987–994. doi:10.1016/S0142-9612(03)00621-5.

28.

Jayakumar

Nagahama

Furuike

and Tamura

, Synthesis of phosphorylated chitosan by novel method and its characterization, Int J Biol Macromol. 42(4) (2008), 335–339. doi:10.1016/j.ijbiomac.2007.12.011.

29.

Goller

Oktar

Agathopoulos

Tulyaganov

Ferreira

Kayali

and Peker

, Effect of sintering temperature on mechanical and microstructural properties of bovine hydroxyapatite (BHA), J Solgel Sci Technol. 37(2) (2006), 111–115. doi:10.1007/s10971-006-6428-9.

30.

Prokopiev

Sevostianov

Genin

McGee

S.M.

and Woodward

, Microstructure and elastic properties of sintered hydroxyapatite, Int J Frac. 130(3) (2004), L183–L190. doi:10.1007/s10704-004-2589-0.

31.

Tang

and Zhou

, Preparation of uniform porous hydroxyapatite biomaterials by a new method, Applied Surf Sci. 254(17) (2008), 5359–5362. doi:10.1016/j.apsusc.2008.02.059.

32.

Kim

Y.-K.

Kim

S.-G.

Yun

P.-Y.

Yeo

I.-S.

Jin

S.-C.

J.-S.

Kim

H.J.

S.K.

Lee

S.Y.

Kim

J.S.

I.W.

Jeong

M.A.

and Kim

G.W.

, Autogenous teeth used for bone grafting: a comparison with traditional grafting materials, Oral Surg Oral Med Oral Pathol Oral Radiol. 117(1) (2014), e39–e45. doi:10.1016/j.oooo.2012.04.018.

33.

Fulmer

M.T.

Ison

I.C.

Hankermayer

C.R.

Constantz

B.R.

and Ross

, Measurements of the solubilities and dissolution rates of several hydroxyapatites, Biomaterials 23(3) (2002), 751–755. doi:10.1016/S0142-9612(01)00180-6.

34.

Pappalardo

Carlino

Brutto

and Sinatra

, How do biomaterials affect the biological activities and responses of cells? An in vitro study, Minerva Stomatol. 59(9) (2010), 445–464.

35.

Motamedian

S.R.

Tabatabaei

F.S.

Akhlaghi

Torshabi

Gholamin

and Khojasteh

, Response of dental pulp stem cells to synthetic, allograft, and xenograft bone scaffolds, Int J Periodontics Restorative Dent. 37(1) (2017), 49–59. doi:10.11607/prd.2121.

36.

Seebach

Schultheiss

Wilhelm

Frank

and Henrich

, Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro, Injury 41 (2010), 731–738. doi:10.1016/j.injury.2010.02.017.

37.

Miron

R.J.

Sculean

Shuang

et al., Osteoconductive potential of a novel biphasic calcium phosphate bone graft in comparison with autografts, xenografts, and DFDBA, Clin Oral Implants Res 27 (2016), 668–675. doi:10.1111/clr.12647.

38.

Naujoks

Langenbach

Berr

et al., Biocompatibility of osteogenic predifferentiated human cord blood stem cells with biomaterials and the influence of the biomaterial on the process of differentiation, J Biomater Appl 25 (2011), 497–512. doi:10.1177/0885328209358631.

39.

Bandyopadhyay-Ghosh

Reaney

I.M.

Brook

I.M.

Hurrell-Gillingham

Johnson

and Hatton

P.V.

, In Vitro Biocompatibility of Fluorcanasite Glassceramics for Bone Tissue Repair, J. Biomed. Mater. Res. A. 80 (2006), 175–183.

40.

Annaz

Hing

K.A.

Kayser

Buckland

and Di Silvio

L.J.

, Porosity variation in hydroxyapatite and osteoblast morphology: a scanning electron microscopy study, J Microsc. 215(Pt 1) (2004), 100–110. doi:10.1111/j.0022-2720.2004.01354.x.

41.

Tabatabaei

F.S.

Tatari

Samadi

and Torshabi

, Surface characterization and biological properties of regular dentin, demineralized dentin, and deproteinized dentin, J Mater Sci Mater Med. 27(11) (2016), 164. doi:10.1007/s10856-016-5780-8.