Physicochemical characterization: Comparative ...

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Original Research Physicochemical characterization: Comparative evaluation of three allograft biomaterials and autogenous bone Antoine Berberi, Antoine Samarani1, Nabih Nader, Rita Mearawi, Wasfi Kanj, Bassam Badran1 ABSTRACT Department Of Oral And Maxillofacial Surgery, School of Dentistry, Lebanese University, 1 Ecole Doctorale, Prase, Lebanese University, Lebanon

Address for correspondence: Dr. Antoine Berberi E‑mail: anberberi@gmail. com

Objectives: Bone substitutes (BSs) used in oral surgery include allografts, xenografts, and synthetic materials that are frequently used to compensate bone loss or to reinforce repaired bone by encouraging new bone in growth into the defect site. The aim of this study was to evaluate a number of physical and chemical properties in a variety of allografts biomaterials used in oral surgery and to compare them with those of autogenous bone. Materials and Methods: Autogenous bone and three different allograft biomaterials were studied by high‑resolution X‑ray diffractometry (XRD), atomic absorption spectrometry, laser diffraction, and checked for their chemical composition, calcium release concentration, crystallinity, and granulation size. Results: The highest calcium release concentration was 24.94 mg/g for Puros® and the lowest one was 4.05 mg/g for OsteoSponge® compared to 20.15 mg/g to natural bone. The range of particles size, in term of median size D50, varied between 630.47 μm for Puros® and 902.41μm for OsteoSponge,® compared to 282.1μm for natural bone. Bone and Puros® displayed a hexagonal shape as bone except and OsteoSponge® which showed a triclinic shape. Conclusion: A BS of choice depends largely on its clinical application that is associated to its biological and mechanical performance. These morphological differences between biomaterials greatly influence their in vivo behavior of biomaterials. Significant differences were detected in terms of calcium concentration, particles size, and crystallinity. KEY WORDS: Allograft, bone, calcium concentration, granulometry, X‑ray diffraction

Introduction

B

one is a living tissue that serves structural support and calcium metabolism. Bone matrix is organic and consists of a network of collagen protein fibers impregnated with mineral salts (85% of calcium phosphate, 10% of calcium carbonate, and 5% of calcium fluoride and magnesium fluoride). The mineral compartment of bone is predominantly present in the form of calcium hydroxyapatites (Ca10[PO4]6[OH]2). Bone tissue also contains negligible quantities of non‑collagen proteins, including the family of bone morphogenetic proteins (BMPs).[1] Access this article online Quick Response Code:

Website: www.jdrr.org DOI: 10.4103/2348-2915.146491

Calcium plays an important role on osteoconductivity and on enhanced bone tissue integration via entrapment and concentration of circulating bone growth factors (BMPs) and osteoprogenitor cells, thus imparting osteoinductive properties of these materials.[2,3] The effect of calcium ions on bone tissue response has been investigated in several in vivo as well as in vitro studies, where Ca‑incorporated titanium implant were shown to enhance osseintegration.[4‑7] Bone tissue serves as a reservoir and a source of calcium for metabolic needs through the process of bone remodeling. The role of phosphate in bone development also should not be over looked since bone maintains body’s pH balance by producing additional carbonates and phosphates. First phosphorus is laid down during the mineralization process, and then calcium binds to it.[3,8] Autogenous bone is osteogenic (cells within a donor graft synthesize new bone at implantation sites), osteoinductive (new bone is formed by active recruitment of host mesenchymal stem cells from surrounding tissue, which differentiate into

How to cite this article: Berberi A, Samarani A, Nader N, Mearawi R, Kanj W, Badran B. Physicochemical characterization: Comparative evaluation of three allograft biomaterials and autogenous bone. J Dent Res Rev 2014;1:132-6. 

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Journal of Dental Research and Review ● Sep-Dec 2014 ● Vol. 1 ● Issue 3

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bone‑forming osteoblasts), osteoconductive (vascularization and new bone formation into the transplant), and highly biocompatible.[3,8] This process is facilitated by the presence of growth factors within the autogenous bone material (mainly BMPs). [9] These characteristics should be present in an idealsubstitute and all bone grafting materials canbe classified according to these characteristics.[10] The greatest success in bone grafting has been achieved with autogeneous bone (“gold standard”), fulfilling all essential physicochemical and biological properties, despite its inherent limitations as availability and postoperative pain.[11,13‑22] Numerous biomaterials have been successfully used as BS, such as allografts (human), xenografts (porcine or bovine), and synthetic calcium‑based materials (β‑tricalcium phosphate (β‑TCP) and hydroxyapatite (HA)), and a combination of these with or without the use of membrane and screws.[11,12,17,23‑25] Allografts do not have the drawbacks of autografts, but are less successful in clinical practice.[26‑29] Ideally, a BSshould be osteoconductive, providing three‑dimensional scaffolds for growth of vessels and osteoprogenitor cells. Finally, it should be resorbable. Moreover, resorption rate should match the formation rate of new bone tissue.[30] In fact; while, a too fast degradation of the biomaterial can exert negative effect on bone regeneration processes,[26] the presence of BS residual grafted particles after bone healing may lead to formation of a “composite repair tissue” rather than to a regenerated bone tissue.[29] The aim of this study was to evaluate some of physical and chemical properties in a variety of commercially available allograft biomaterials that are frequently used for dental applications as BS and to compare them with autogenous bone.

Materials and Methods A total of three commercially available allograft BS were included in this study (Puros®, OsteoSponge®, and DynaBlast™). Each material was used with the lowest particle size range available. Analytical methods were used to investigate these properties, techniques such as atomic absorption spectrometry (AAS), laser diffraction (LD), and X‑ray diffractometry (XRD). AAS was used to determine concentration of calcium ion in BS, LD was used to measure granulate size, and XRD to identify phase and composition features and qualitatively evaluate crystallinity of materials. Comparisons of the properties of the commercial products with the properties of autogenous bone were also explored. The following data were taken from the manufacturer’s specifications. All samples were obtained directlyfrom Journal of Dental Research and Review ● Sep-Dec 2014 ● Vol. 1 ● Issue 3

manufacturers in sealed vials and used without further treatment. DynaBlast™ (Keystone Dental, Inc, 144 Middlesex Tumpike Burlington, MA 01803, USA) is a combination of human tissue (tissue banks in the USA) that has been demineralized and cancellous bone mixed with poloxamer reverse phase resorbable medium and formulated into a paste or putty‑like form.[31] Puros® (Zimmer Dental Inc, 1900 Aston Ave. Carlsbad, CA 92008, USA) is an allogeneic graft material treated by Tutoplast® process that gently removes unwanted material such as fat, cells, antigens, and inactivates pathogens; while preserving the valuable minerals and collagen matrix. It is available as cortical granulate, particle size 0.25–2 mm.[28,32] OsteoSponge® (Bacterin International Inc, 600 Cruiser Lane Belgrdae, MT 59714 USA) allograft consists of 100% of demineralized human cancellous bone, with no additional carrier materials. OsteoSponge® is prepared using methods that preserve native growth factors.[27] The granulate size varies between 1 and 4 mm. The bone samples were collected during mandibular third molars surgery (gamma‑irradiated), rinsed with ethanol, dried in vacuum at room temperature, and ground in agate mortar.[33,34]

AAS Calcium and phosphorous release from graft material in demineralized water was evaluated using AAS (WFX‑210, RayLeigh, BRAIC, China). For this aim, standards for calcium and phosphorous within the range between 0.5 and 10 µg/L were prepared. 0.4mg of each biomaterial (all nine sample) was immersed in100mL of 0.9% NaCland the pH was adjusted at 7 by using hydrochloric acid (0.1N). The variation of Ca concentration was determined at D0 (day 0), D2 (day 2), and each week after, until the week 6. The concentration was calculated based on the Beer–Lambert law.[35]

LD for particle size measurement Particle size distribution was determined using a laser scattering particle size analyzer (Patrica LA‑950 V2 Horiba Instruments, Japan). The measurement method relies on the basis of Mie scattering theory, with measuring range varying between 0.01 and 3 μm using an ultrasonic probe with measuring time of 20s at a frequency of 20 kHz. The devise is equipped with an optical system of two light sources, a laser diode of approximately 1.6 mW with λ = 650 nm and a 405nm light‑emitting diode of approximately 0.3 mW. Large particles scatter light at small angles relative to the laser beam and small particles scatter light at large angles. The particle size is reported as a volume equivalent sphere diameter.[36,37] Samples were well mixed and homogenized in their powder state prior to their analysis. Average particle size and distribution were calculated for all nine biomaterials studied and autogenous bone. 133 


XRD In order to determine the crystal phases in the explored biomaterials, X‑ray powder diffract meter was used. Homogenized powder samples (2–3 g) were compressed in PVC lenses (diameter 2.5 cm, thickness 2 mm) and measured using a D8 Bruker diffract meter (copper anticathode λ Ka =0.154060 nm). Range of 2θ between x° and y° was chosen to obtain maximum information about crystal phases. Collected diffract grams were analyzed by the software EVA (Bruker Corporation, Billerica, Massachusetts, USA) based on powder diffraction files provided by the International Center for Diffraction Data (ICDD). Crystallite size analysis was calculated using the peak broadening of XRD reflection that is used to estimate the crystallite size (in an orthogonal direction to the crystal plane) using the following formula:[38] Xs=0.9λ / ( FWHM xcosθ ) Where, X sis the crystallite size in nanometer, λ is the wavelength of X‑ray beam in nanometer (λ =0.15406nm in our case), and FWHM is the full width at half maximum for the diffraction angle at 2θ =25.9° that was selected according to (002) Miller’s plane family).

Results AAS results of calcium concentration over the observation period are showed in Table 1. OsteoSponge ® revealed the lowest calcium release concentration (4.05 mg/g) followed by DynaBlast™(6.2 mg/g). The calcium concentration of Puros ® (24.94 mg/g) was comparable to autogenous bone (20.15 mg/g).

The results of the XRD experiments that are indicative for the chemical composition of the BS are shown in Table 3 except for DynaBlast™ as the material is presented in putty paste form and not granulate one. These materials diffract more and less the X‑ray, which means diverse degrees of crystallinity, as indicated in the different peaks widths [Figure 1]. The common crystal phase was calcium phosphate silicate hydroxide (Ca 5(PO 4) 2.85(SiO 4) 0.15(OH)) and autogenous bone. Except for OsteoSponge® samples were crystallized at different levels of crystallinity in hexagonal systems. The OsteoSponge® sample is the only sample crystallized in triclinic system.

Discussion The more higher the calcium concentration, more are the biomaterials prone to degradation. [3,33,34] The acidic buffer, to some extent, mimics the acidic environment during osteoclastic activity or bone resorption.[3,4,33] In vivo biodegradability can be achieved by dissolution or cell‑mediated, multinuclear cells; osteoclasts; and macrophages are involved in phagocytosis.[3,33,34,37,39] In our study, different allograft biomaterials had different calcium releasing. This could be explained by the fact that the speed of biodegradability of BS, in vivo or in vitro, depends Table 1: The calcium concentration as derived from AAS experiments by brand names and time period Ca (mg/g)

Puros®

OsteoSponge®

DynaBlast™

Bone

2.104 3.613 6.99 15.1535 18.879 23.11 24.942

1.521 1.723 1.859 2.018 2.18 2.493 4.051

2.768 3.7132 4.871 5.6507 5.8985 6.0485 6.2

3.77 4.2 6.73 16.4 17.96 19.64 20.15

The particles median size D50 (in volume percentages), the particle size range expressed by the 10 and 90 percentiles (D10 and D90, respectively) and the particles size ranges reported by the manufacturers as determined by the LD measurements are all presented in Table 2.

Day 0 Day 2 1 week 3 week 4 week 5 week 6 week

Results showed that Puros® had the lowest median particle size (630.47 μm) followed by DynaBlast™ (777.14 μm), while OsetoSponge® had the highest one (902.41μm).

AAS: Absorption spectrometry

No one shows a median size close to the one ofautogenous bone (282.1 μm). The narrowest size distribution was observed with BioOss ® (0.26–8.92μm) followed byIngenios ™β‑TCP (3.90–15.18 μm). The widest size distribution was observed with OsteoSponge ® (174.62–2,301.84 μm) followed by DynaBlast ™ (39.24–1,754.62 μm) and Puros ® (174.62– 1,167.72 μm).

Table 2: Particle size parameters in volume percentage of the samples Sample

Median size (D50 μm)

Size range (D10-D90 μm)

Size range reported By producers (μm)

630.47 902.41 777.14 282.1

174.62-1,167.72 152.45-2,301.84 39.24-1,754.62 90.5-465.15

250-2,000 1,000-4,000 Not indicated

Puros® OsteoSponge® DynaBlast™ Autogenous bone

Table 3: Chemical compositions and shapes of samples as derived from X‑ray diffractometry except for DynaBlast® (presented in putty paste from) Product

Compound name

Formula

Shape

a (Å)

b (Å)

c (Å)

Alpha

Beta

Gamma

Puros OsteoSponge® DynaBlast™ Autogenous bone

Calcium phosphate silicate hydroxide Calcium phosphate silicate hydroxide

Ca5(PO4)2.85(SiO4)0.15(OH) Ca5(PO4)2.85(SiO4)0.15(OH)

Hexagonal Triclinic

9.42 6.25

9.42 11.9

6.89 5.6

90 97

90 114

120 93

Calcium phosphate silicate hydroxide

Ca5(PO4)2.85(SiO4)0.15(OH)

Hexagonal

9.42

9.42

6.89

90

90

120

®



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Figure 1: X-ray diffraction data for all investigated samples. All data were measured at. 1.54 Å as wavelength and the displayed range in 2Ø was chosen to optimally represent the relevant features

on their composition, particle size, crystallinity, porosity, and preparation.[3,33,34,37,39] From the particle size related data, it can be concluded that, in general, size ranges measured for tested materials were different from those reported by manufacturers who do not specify the used technique in the crystalline material’s characterization and could explain the noticed differences.[40‑44] However, it should be kept in mind that the granules under analysis differed not only in their size but also in their physicochemical properties. The influence of properties and characteristics of BS on biological response cannot be easily predicted as the published studies involve different types of BS in different particle size ranges. Regarding the ranges of particle size that were tested in the present investigation, there was no relation between the sizes of particles and calcium concentration with the time. XRD diffractograms of various materials, including human bone, were quite similar with common crystal phase calcium phosphate silicate hydroxide (Ca5(PO 4) 2.85(SiO 4) 0.15(OH). However, silicates, when they are present, are not major components of the crystal phases, since their stoichiometry compared to phosphate (2.85) are considerably negligible (0.15). It must be highlighted that in bone and all BS Ca to P ratios are fluctuating between 1.75 and 1.33. This could mean that calcium is the major element that compensates phosphate charges. XRD diffractograms showed that all samples, including natural bone, proved to have the same anisotropic crystal size (9.42Å in a and b‑directions and 6.8Å in c‑direction with alpha and beta 90° and gamma 120°), OsteoSponge® showed different Journal of Dental Research and Review ● Sep-Dec 2014 ● Vol. 1 ● Issue 3

anisotropic size 6.2Å in a and 11.9Å in b‑directions and 5.6Å in c‑direction with alpha and beta 97° and gamma 120°; 6.25Å in a‑direction, 11.9 Å in b‑direction, and 5.6 Å in c‑direction with alpha 97 and beta 114° and gamma 93°, respectively. Theseresults demonstrated that crystal shape of Puros®and autogenous bone are similar, hexagonal shape, and OsteoSponge® showed a triclinic shape. This structure is the poorest system in the symmetric properties.

Conclusions Three different allograft materials were herein investigated, and results were compared to autogenous bone. Even when similar chemical characteristics were found, significant differences were detected in terms of calcium concentrations, particles size, and crystallinity. Although these morphological differences greatly influence in vivo behavior of the biomaterial, they are often not taken into consideration when the samples’ biological performance is evaluated. It is believed that results provided for biomaterials investigated will be most useful to fully understand their clinical behavior and response. Since, the BS of choice depends largely on the possible clinical application and its associated biological and mechanical needs, it is important not to assume that all BS will show the same pattern of performance and that the validation of a BS in one clinical site may not necessarily predict its identical performance in another anatomical location. Hopefully in the future, hybrid or complex combination products that include cells, growth factors, and/or gene therapy in combination will be likely to provide oral surgeons more effective tools for bone defects reparation. In this regard, it is obvious that further studies are warranted. 135 


Acknowledgments The authors would like to thank Manal Houhou, Sarah Hadda, Sahar Rihan, and Hussein Bassal for their excellent technical assistance.

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Source of Support: This project was supported by a grant from the EcoleDoctorale, Lebanese University, Conflict of Interest: None declared.
