Evaluation of Diammonium hydrogen phosphate and Ca(OH)2 nanoparticles for consolidation of ancient bones

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Original article

Evaluation of Diammonium hydrogen phosphate and Ca(OH)2 nanoparticles for consolidation of ancient bones Annalisa Salvatore a , Stefania Vai b , Stefano Caporali c , David Caramelli b , Martina Lari b,∗,1 , Emiliano Carretti a,∗ a

Department of Chemistry “Ugo Schiff” and CSGI, University of Florence, Firenze, Italy Department of Biology, University of Florence, via del Proconsolo 12, 50122 Firenze, Italy c Department of industrial Engineering (DIEF), University of Florence, via di S. Marta 3, 50139 Firenze, Italy b

a r t i c l e

i n f o

Article history: Received 8 April 2019 Accepted 30 July 2019 Available online xxx Keywords: Nanoparticles Aragonite Hydroxyapatite Ancient DNA Bone remains

a b s t r a c t The development of innovative conservation strategies for the restoration and conservation of bone finds of historical and archaeological interest is crucial to warrantee the fruition of the relics and, most important, to maintain the long-term access to the biological information recorded in the bones. The approach purposed in this study is based on smart nanostructured inorganic materials with both high physical and chemical compatibility with the treated support. In particular, an aqueous solution of diammonium hydrogen phosphate (DAP), which is a precursor agent for the growth of a 3D crystalline network of hydroxyapatite (HAP) has been used for the consolidation of degraded ancient bones of historical and archaeological interest to restore a continuous crystalline HAP network in the regions of the bone relics affected by degradation phenomena. The consolidation performance of the HAP has been evaluated also after a pre-treatment with Ca(OH)2 nanoparticles (NPs), successfully used in the last years for the consolidation of both carbonatic matrixes of artistic interest and of bone relics. The treated bone relics have been characterized in terms of porosity and surface area through gas porosimetry measurements: the in-situ precipitation of hydroxyapatite with or without pre-treatment with Ca(OH)2 NPs affects the overall porosity and the adsorption-desorption isotherms obtained through the Brunauer Emmet and Teller (BET) theory indicate a decrease of surface area and pore volume of 45% and 64% respectively. Xray microtomography images clearly show, after the treatment, the formation of a homogeneous dense phase, confirming the consolidating effect on the bone relics. Moreover, it has been also observed that the consolidation treatment induces an increase of the Vickers microhardness higher than the 40%. Additionally, by means of a paleogenetic analysis we showed that authentic genetic data could be retrieved from bones even after the consolidating treatments. © 2019 Elsevier Masson SAS. All rights reserved.

1. Introduction Bones recovered from archaeological excavations are not simply objects of historical and archaeological interest. They represent a true “Biological Archive” that deserves to be available and accessible for long-term studies. Particularly, ancient human bones represent an important resource for understanding early human societies and past economies. Human skeletal remains offer the most intimate link to our ancestors, providing important evidence on belief systems, burial rituals and social hierarchies, as well as

∗ Corresponding authors. E-mail addresses: martina.lari@unifi.it (M. Lari), emiliano.carretti@unifi.it (E. Carretti). 1 http://www.invalid.uri/.

contributing to our understanding of diet, disease and genetic affiliations in the distant past [1–5]. Similarly, archaeological animal bone assemblages represent a powerful resource in the understanding of past environments and the exploitation of natural resources by earlier societies, as well in reconstructing animal domestication processes [6–8]. Chemically, a bone is a composite material composed by water (10 wt%), collagen (20–30 wt%) and minerals (60–70 wt%) [9]. The mineral component is made mainly by apatite, having general formula Ca10 (PO4 )X2 , with X usually representing hydroxyl (hydroxylapatite, HAP) or low amounts of fluorine, or chlorine ions (fluoroapatite and chloroapatite respectively). Moreover, low amounts of magnesium and strontium ions substitute calcium into the crystalline network. The HAP crystals are very small and arranged in a sort of 3D microcrystal mosaic texture, rather than well-developed single crystal structures [10]. For an archeological bone, in a thermodynamically open

https://doi.org/10.1016/j.culher.2019.07.022 1296-2074/© 2019 Elsevier Masson SAS. All rights reserved.

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environment, such as the soil of a burial, the preservation level is strictly related to the local physico-chemical parameters such as the pH of the soil, soil moisture, environmental redox potential, microbial activity and temperature. Moreover, it is worth considering that usually well conserved archaeological bones have a porosity ranging between 20 and 30% by volume, almost half of which due to pores having a radius below 4 nm; as a result, these matrixes are characterized by a high extension of their interface that is available for exchange of matter and energy with the external environment. All these factors simultaneously contribute to the chemical deterioration and biodegradation of collagen and of the mineral phases [11–13]. At today, and since the start of the twentieth century, the products that are extensively used for the consolidation of bone remains mainly include natural and synthetic organic polymers (vinyl and acrylic polymers and copolymers, alkoxysilanes, cellulose derivatives, agar, polyethylene, etc.) [14]. Unfortunately, the extensive use of these materials causes many problems mainly due to their scarce chemical stability that results in simultaneous depolymerization and crosslinking reactions. Then the weathering of these consolidation materials first rapidly leads to an alteration of their consolidation performances; moreover, their deterioration also results in yellowing of the treated matrix, in shrinking phenomena that can induce mechanical stresses to the matrix in which they are applied (increasing the embrittlement that result in delamination and formation of local detachments and gaps) and in a strong decrease of their solubility that reduces the reversibility of the treatment (that is mandatory if the consolidation performances are completely or even partially compromised) up to the 50% [14]. Moreover, in addition to these damages they can also limit the possible re-treatability of the object and future analysis such as radiocarbon dating, isotope analysis and the access to the residual genetic heritage. Despite all these problems related to the application of acrylic and vinyl polymers and copolymers, at today they are still the most diffused products for the consolidation of bone remains. The development of more proper and affordable strategies for the restoration and conservation of ancient bones is therefore crucial to warrantee the fruition of the relics and, most important, to maintain long-term access to the biological information recorded in the bones. A possible effective alternative to organic polymers has been presented in a previous paper [15] where Ca(OH)2 nanoparticles (NPs) have been successfully used for the first time as consolidating agent for bone remains. Through the application of this innovative method based on the use of nanotechnologies, it is possible to strongly increase the physico-chemical compatibility between the consolidating agent and the porous network, being the bones mainly composed by an inorganic mineral matrix. Ca(OH)2 NPs have been specifically developed for the deacidification of cellulose based materials [16,17] and to consolidate carbonate matrices (i.e., wall paintings, carbonatic stones) affected by surface degradation phenomena [18–20]. In the latter case, once applied, Ca(OH)2 NPs react with atmospheric CO2 repristinating the original texture by forming a 3D network of new crystals of CaCO3 . Moreover, it has been also demonstrated [15] (and references therein) that the collagene in the presence of Mg2+ , both these substances are naturally present into a bone relic, can promote the in-situ formation of aragonite, through the carbonation of Ca(OH)2 NPs by means of the atmospheric CO2 . This mineral, due to its high hardness (2.25 GPa vs. 1.54 GPa of calcite) should favor a strong increase of the strength of the consolidated relic (up to 200%) together with an increase of the mineral density of the bone matrix and a decrease of the porosity. In this paper, we present a highly innovative restoration technique to address the conservation (mainly long-term

strengthening) of more or less decohesive bone finds of historical, archaeological and even paleontological interest. The way that has been followed for the first time involves the development of an innovative consolidation approach based on the use of highly compatible and performing inorganic materials. The major novelty of this paper is the multidisciplinary approach purposed that involves Chemistry and Biology: from one side it covers the development of innovative materials in response to real conservation needs and from another the application of the novel materials has been evaluated for the first time with particular attention to their impact on the preservation and recovering of endogenous DNA still present inside the bone finds. In order to increase both the durability of the consolidation treatment and the physio-chemical compatibility between the matrix and the consolidating agent, a possible approach is based on the use of consolidants constituted by inorganic materials. Inorganic precursors including calcium hydroxide both as dispersions and colloids [21,22], barium hydroxide and oxalate salts, have been tested and used as alternatives to organic polymer-based reagents for the consolidation of porous materials, mainly stones and plasters. Recent studies, mainly focused on the consolidation of stone materials of historical and artistical interest by bio-mimicking the growth of HAP inside of the stone porous matrix, have demonstrated considerable potential of this compound as consolidant and/or protective for carbonatic stone artifacts [23–25]. These preliminary applications in the field of conservation, together with significant fundamental research focused on the precipitation of HAP for bioengineering and biomedical applications [26,27], suggest the use of HAP as a consolidating agent for degraded archeological bones. In order to improve the chemical compatibility with the matrix of the bone respect to the traditional procedures commonly used for their consolidation that are based on the use of polymers, the attention has been focused on the possibility to consolidate bone remains by inducing the precipitation of hydroxyapatite (HAP) directly into the porous matrix of the bone by using diammonium hydrogen phosphate (DAP) as precursor of HAP [28,29]. Hence, as a matter of principle the action of the HAP could be coordinated with the one of Ca(OH)2 NPs in order to maximize at the same time both the improvement of the strength of the porous support and the compatibility of the consolidation treatment with the bone matrix. In that way, the CaCO3 particles formed should act as filler for the larger cavities and they can represent a reservoir of calcium that, through its reaction with the added DAP can promote the crystallization of new HAP and form a tightly interconnected structure. One of the most important novelties introduced with this paper, besides the use of a high durable and high compatible consolidation product, is that for the first time the evaluation of the performances of a consolidation treatment of a bone of historical and archaeological interest has been carried out not only considering its impact on the physico-chemical properties of the treated support, but mainly considering the effect on the analysis of genetic material. Then, it has been investigated how the consolidation treatment influences the access to the paleogenetic information respect to a not consolidated bone. In order to perform a preliminary evaluation of the impact of consolidation on molecular analysis of ancient skeletal materials, we followed a Next Generation Sequencing (NGS) approach coupled with target capture enrichment for recovering complete mitochondrial genomes from a small set of archaeological bones. We applied an experimental workflow specifically developed for recovering DNA from ancient and degraded biological materials and used bioinformatics tools to reconstruct and validate the genetic data obtained from both consolidated and untreated bone fragments.

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2. Research aim Set up of an innovative and chemically compatible procedure for the consolidation of bone remains of historical and archaeological interest based on the use of inorganic materials such as diammonium phosphate and Ca(OH)2 nanoparticles. Evaluation of the effects of the treatment on the physico-chemical properties of the bone relics (porosity, pore size distribution, surface area and hardness). Evaluation of the effects of the treatment on the genetic heritage. 3. Materials and methods 3.1. Materials Calcium hydroxide nanoparticles dispersed in 2-propanol (0.5 g/L) were prepared by a size and shape controlled synthesis at the CSGI laboratories, and used without further purification. Diammonium hydrogen phosphate (NH4 )2 HPO4 (DAP) has been purchased by Fluka Sigma Aldrich. The tests have been carried out on two different series of real archaeological human bone remains (Fig. 1). Bones fragments with comparable size and morphological preservation were selected. Additional sampling criteria were the absence of previous restoring or consolidant treatments, and the preliminary evidence that adequate amount of endogenous DNA was still preserved in the bones. Fragments of a long bone (femur) belonging to an Iron Age individual from Italy (Fig. 1a) were used to evaluate the impact of the consolidation treatment on the physico-chemical properties that have been analyzed before and after the consolidation. An additional set of samples was selected for testing the effects of consolidants on the quality of molecular genetics data that can be recovered from the ancient bones. Bone samples come from a Neolithic burial in Poland, Kierzkowo, attributed to the Globular Amphorae culture. Four radiocarbon dates are available for the site and indicate a chronology between 4100 and 4450 BP [30]. Anthropological and genetic data from previous studies are available [31,32]. The samples analyzed here consists of fragmented long bones from three different human

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individuals labeled as K3.4 (left femur), K5.3 (right femur), and K6.2 (right ulna) (Fig. 1b). Considering that several analytical techniques were applied to deeply test the effects of two different consolidation treatments (see below), the choice of using different samples for physico-chemical and genetic evaluations was mainly driven by the need of preserving the different bone remains.

3.2. Bone consolidation The bone samples have been first cleaned by gentle brushing. Using a circular portable saw three small pieces were obtained from each bone: one piece was left untreated (NT sample) and the others were subjected to consolidation treatment 1 and consolidation treatment 2. In order to maximize the consolidating effect, the bone samples have to be completely soaked with consolidant solutions and dispersions. This goal could be achieved by brushing or spraying these products until saturation or by immersion, as a function of the dimensions and of the operative conditions. In this case, being the dimensions of our samples in the order of few centimeters, the two different consolidation procedures that have been tested (Scheme 1), are based on the immersion of the samples into the consolidant solutions and dispersions. They are indicated as consolidation 1 and consolidation 2 as follows: • consolidation 1: the samples were soaked by immersion in an aqueous solution 1 M of diammonium hydrogen phosphate (NH4 )2 HPO4 (DAP), used as the precursor for the precipitation of HAP. After that, the samples were maintained for two weeks in a confined environment at RH = 75% before evaluation; • consolidation 2: the samples were first soaked by immersion in a 0.5 g/L of Ca(OH)2 NPs dispersed in 2-propanol for 2 hours, then the samples were allowed to dry under hood at RT for one week. The second step involved the soaking by immersion in an aqueous solution of DAP 1 M in deionized water for 2 hours, then the samples were maintained for two weeks in a confined environment at RH = 75% before evaluation.

Fig. 1. Human bone remains selected for evaluating the consolidants. Fragment of femur from an Iron Age individual used to evaluate the impact of the consolidation treatment on the physico-chemical properties of bones (a). Fragmented long bones from the Neolithic site of Kierzkowo used for testing the effects of consolidants on the quality of molecular genetics data recovered from the bones (b).

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Scheme 1. Representative sketch describing the consolidation treatments on the bone remains. Consolidation 1: involved only one soaking step in an aqueous solution of DAP 1 M. Consolidation 2: it was based on a first soaking in 0.5 g/L Ca(OH)2 NPs dispersion in 2-propanol for 2 hours, followed by a completely drying under hood for one week, then a second soaking was done in an aqueous solution of DAP 1 M for 2 hours, the following two weeks the sample was maintained in a confined environment at RH = 75%. Acronym NT indicates the untreated bone.

3.3. Analytical techniques 3.3.1. X-ray diffraction Powder X-ray diffraction (XRD) measurements were carried out on powdered bone samples (ca 300 mg each) to detect the formation of new crystalline phases induced by the application of the consolidants. XRD spectra were collected at CRIST Centre, University of Florence (Italy), using a Bruker D8 Advance diffractometer equipped with a Cu K␣ radiation and operating in ␪-2␪ BraggBrentano geometry at 40 kV and 40 mA. 3.3.2. X-ray microtomography ␮-CT data were collected on bone fragments (their dimensions range in the order of the hundreds of ␮m) in order to evaluate the effect of the consolidation treatments on the mineral density of the bones. Measurements were carried out using a Skyscan 1172 highresolution MicroCT system at CRIST Centre, University of Florence (Italy). This system has a sealed, microfocus tungsten X-ray tube with a 5 ␮m focal spot size. The X-rays were produced by exposing the anode to an electron beam at 59 kV and 167 ␮A. Each sample was placed on the pedestal between the X-ray source and the CCD detector and the 2D X-ray images were captured over 180 rotating sample with a slice-to-slice rotation angle of 0.3, each 2D image represents one slice. The total acquisition time was approximately 40 minutes. Spatial resolution of the image was kept in a range of 1 ␮m. The 3D image stacks are reconstructed from the rotation image projections, using the reconstruction software (Bruker provides the NRecon software). NRecon software allows the adjustment of three reconstruction parameters: smoothing, beam-hardening factor correction, and ring artefact reduction. After reconstruction, the images can be analyzed to obtain quantitative information on the bones structure (CTAn software). An identification of phases was performed by observing 3D images extracted from the reconstructed volumes of samples and coloring them using CTVol software. 3.3.3. FT-IR measurements FTIR spectra were collected on powdered bone samples (few mg each) before and after the consolidation to detect the formation of new chemical substances after the consolidation and to evaluate the chemical compatibility of the consolidation treatment with

the bone matrix. Fourier transform infrared spectroscopy measurements were carried out using a BioRad FTS-40 spectrometer in the range 4000–400 cm−1 . Spectra are averages of 128 scans recorded in absorbance mode with 2 cm−1 resolution. KBr pellets were prepared by finely grinding and mixing 40 mg of bone samples with 250 mg of pure KBr. 3.3.4. Scanning electron microscope A FEG-SEM IGMA (Carl Zeiss, Germany) was used to collect micrographs of some bone fragments (their dimensions ranged in the order of few hundreds of ␮m) in order to evaluate the effect of the consolidation treatments in terms of morphology of the bone surface. The SEM images were collected using an acceleration potential of 1 kV and a working distance of 1.4 mm. 3.3.5. Gas porosimetry Nitrogen adsorption and desorption isotherms were acquired to obtain information on the effect of the consolidation treatments on the overall porosity and on the pore size distribution of the bone samples. The measurements were carried out using a Beckman Coulter SA-3100 Surface area analyser. Bone samples (2–3 g of bone fragments having dimensions in the order of few mm) were outgassed prior to analysis in vacuum conditions at a temperature of 40 ◦ C until a pressure of 0.01 mmHg was reached. The outgas temperature was chosen in order to avoid hydrolyzation of collagen. 3.3.6. Ion chromatography Ion chromatographic analysis was carried out to evaluate the presence of Mg2+ ions in the bone matrix. This was performed using a DionexDX120 ionic chromatograph equipped with a Dionex CS12A column and using Na2 CO3 /NaHCO3 2.5 mM as eluent. 3.3.7. Vickers Microhardness Measurements Microhardness measurements were carried out to study the effect of the consolidants on the mechanical properties of the bones. The bone microhardness was measured at room temperature by means of a HX-1000 TM (Remet s.a.s., Italy) tester using a Vickes square base indenter and applying 25 g for 15 s in order to let the system to equilibrate. The images were analyzed by means of AUTOVICKERS software. Microhardness values were obtained by averaging 10 measurements obtained on randomly chosen portions

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of the sample surface (the dimensions of the samples are in the order of 1–2 cm). 3.3.8. Color measurements The colour measurements were carried out by means of X-RITE SP60 VIS portable spectrophotometer in order to evaluate any color change induced by the consolidation treatments. The color change E was given by: ∗ E12 =





L2∗ − L1∗

2



+ a∗2 − a∗1





2



+ b∗2 − b∗1

where L1∗ , a∗1 , b∗1 and L2∗ , a∗2 , b∗2 nates of two different colors.



2

are the CIELAB coordi-

3.3.9. Paleogenetics analysis In the Laboratory of Molecular Anthropology and Paleogenetics, small bone fragments (their dimensions are in the order of 1 × 2 cm) were obtained from each of the three specimens from Kierzkowo using diamond wheels mounted on a dental device (MarathonMulti 600 Micromotor). We had care to produce fragments with similar features regarding dimension, thickness and bone matrix. For each sample, two fragments were moved to the Laboratory of the Chemistry Department of the University of Florence where the consolidating methods previously described were applied. A third fragment did not receive any consolidation treatment and was used as control for DNA analysis. After consolidation treatments, the bone fragments were moved again to the Laboratory of Molecular Anthropology and Paleogenetics and randomly processed alongside the untreated fragments to avoid any experimental bias. The bone surface was cleaned with disposable rotary tools mounted on the same dental device used for cutting bones (Marathon-Multi 600 Micromotor), then exposed to UV lights at 254 nm in a UVLink Crosslinker for 45 minutes. Bone powder (approximately 50 mg) was collected from the inner part of the sample using the same disposable rotary tools and 60 mg were used for DNA extraction following a protocol optimized for recovery of short molecules [33]. DNA libraries were performed starting from 20 ␮l of DNA extract without any UDG treatment to allow the analysis of the original patterns of misincorporation [34]. Negative controls were processed alongside the samples to

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monitor modern DNA contamination occurring during experimental procedures. Libraries were enriched for mitochondrial genome (mtDNA) using DNA probes obtained from PCR products [35]. The enriched libraries were pooled together in equimolar amounts and sequenced on a Illumina MiSeq for 2 × 75 cycles. Raw sequence data were analysed using a pipeline specific for ancient DNA samples [36]. Adapters were removed and paired-end reads with a minimum overlap of 10 bp were merged using Clip&Merge version 1.7.4. Merged reads were mapped on the revised Cambridge Reference Sequence, rCRS (GenBank Accession Number NC 012920.1) by CircularMapper in order to take into account the circularity of the mitochondrial genome. Duplicates were removed using DeDup, a tool that considers both ends of the fragments to recognize them as clonal. Reads with mapping quality below 30 were discarded. Mapping reads were analyzed for length and deamination patterns using MapDamage 2.0 [37]. ContamMix [38] was used for contamination estimation. MtDNA haplogroup assignment was performed with HaploGrep [39]. 4. Results and discussion A aqueous solution of DAP 1 M has been used in this study for the consolidation of Iron Age human bones (immersion for 2 hours in the DAP solution) in combination (consolidation 2) or not (consolidation 1) with a dispersion of Ca(OH)2 NPs 0.5 g/L in 2-propanol (Scheme 1). In consolidation 2 Ca(OH)2 NPs have been added in order to promote the crystallization of aragonite by exploiting, during the carbonation reaction induced by the atmospheric CO2 , the simultaneous templating action of collagen and Mg2+ ions24 naturally contained into the analyzed Iron Age archaeological bones (the presence of collagen is indicated by the FTIR spectra reported in Fig. 2 and the concentration of Mg2+ ions is around 1000 ppm as indicated by ion chromatography data). Moreover, several studies regarding denaturation of collagen have demonstrated that acidic and/or alkaline environment can affect the fibrils arrangement, changing their stability and solubility [40,41]. Concerning the consolidation with Ca(OH)2 NPs, the presence of 2-propanol instead of water as liquid medium and the faster kinetics of the carbonatation reaction compared to collagen hydrolysis process [42] allow us to exclude any deleterious effect on the nature of collagen.

Fig. 2. FTIR spectra of a microsample of the Iron Age bone fragment. The two stars indicate the peaks at 1650 cm−1 and 1540 cm−1 due respectively to amide I and amide II of the collagen contained into the bone.

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Fig. 3. XRD patterns collected onto untreated bone sample (NT, a), onto a bone sample subject to consolidation 1 (aqueous solution of DAP 1 M, b) and onto a bone sample subject to consolidation 2 (pre-consolidation with Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M, c). All bone samples belong to an Iron Age individual. Black stars indicates the typical peaks of nanocrystalline hydroxyapatite and the cross indicate biphosammite.

Regarding the consolidation with the DAP aqueous solution, although the pH of the solution is 8, Hattori et al. [43] showed that at this pH value collagen retain the triple helical conformation maintaining its property as a biological adherent molecule. Two weeks after the consolidation, both untreated and consolidate samples were analyzed by means of several technique in order to verify the efficacy and the impact of the consolidation in terms of overall morphology and physico-chemical properties. The consolidation effect on bones subjected to consolidation 1 (immersion up to saturation in an aqueous solution of DAP 1 M) and consolidation 2 (a pre-consolidation with Ca(OH)2 NPs 0.5 g/L in 2-propanol and immersion up to saturation in an aqueous solution of DAP 1 M) was first monitored through color measurements that indicated that the E induced by the consolidation treatment was lower than 2, a value that corresponds to a color change not perceivable by the human eye. Moreover, XRD diffraction, FTIR, gas porosimetry, microtomography and SEM measurements were also carried out.

polymorphs of CaCO3 like calcite into the bone sample subjected to consolidation 2, FTIR analysis was carried out on fragments of untreated and consolidated bone samples; the spectra are shown in Fig. 4.

4.2. FTIR analysis The spectrum of the bone subject to consolidation 2 with a dispersion of Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M (Fig. 4c) shows a peak at 810 cm−1 (due to the out-of-plane bending mode of the O C O bond of the CO3 2− anion) and a peak at 690 cm−1 (in-plane bending mode of the O C O bond of the CO3 2− anion) that are attributable to the presence of aragonite [46]. This is also confirmed by the SEM micrograph reported in Fig. 5 where the presence of needle-shaped crystals, a morphology typical of aragonite, is evident on the surface of the bone subject to consolidation 2.

4.1. XRD analysis XRD patterns obtained from the powder samples of the Iron age bone (Fig. 1) are reported in Fig. 3. The main effect of the application of the consolidants, besides the increase for both consolidated samples of nanocrystalline hydroxyapatite, highlighted with the broadening of the typical diffraction peaks identified with black stars, in the c spectrum (consolidation 2, pre-treatment with Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M, c), only in the spectrum c we also notice the diffraction peak of biphosammite (peak at 23 degrees), which is mainly composed by monoammonium hydrogen phosphate, probably originated from the DAP used for the consolidation [44]. Then, beside the formation of biphosammite induced by the consolidation treatment 2, the chemical composition of the bone matrix is not affected by both the consolidation treatments. Moreover, in the sample subject to the consolidation 2, it is interesting to notice the absence of diffraction peaks attributable to both calcite and aragonite. The reason neither aragonite nor calcite signals were detected in the diffractogram is probably due to their low concentration, below the detection limit, that as indicated by Kontoyannis et al. [45]. for calcite and aragonite is equal to 0.9 w/w% and 2.6 w/w% respectively. Then, in order to detect the presence of aragonite or other

4.3. Gas porosimetry measurements The effects of the treatments in terms of the overall porosity were assessed by gas porosimetry. Nitrogen gas sorption is the most widely and accurate method for total surface area measurements and pore sizes within the approximate range of 0.4 to 200 nm in diameter. Nitrogen adsorption-desorption isotherms were recorded for bone samples powder before and after consolidation treatments (Fig. 6a and b). According to the classification made by IUPAC [47] all analysed samples exhibit a type IV(a) isotherm, characteristic of mesoporous materials ranging from 2 to 50 nm in pore size. Three well-defined regions in the isotherms can be identified for both consolidation treatments (Fig. 6a and b grey lines): • a reduction of nitrogen uptake at very low relative pressure values (P/P0 up to 0.02), that can be associated to micropore filling and monolayer formation; • an inflection point and slope change followed by a more linear region in the isotherm curve confirming reduction of available area for multilayer adsorption;

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Fig. 4. FTIR spectra of the microsamples of the Iron Age bone fragments before the consolidation treatment (NT, spectrum a), after the consolidation 1 (aqueous solution of DAP 1 M, spectrum b) and after the consolidation 2 (pre-treatment with Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M, spectrum c).

The graphs in Fig. 6c and d report the differential pore size distribution (that indicates which pore volume is ascribed to a family of pores having a given pore size) obtained from desorption of gas; although the model applied considers the presence of cylindrical pore [48], which is not our case, it is possible to compare the effect of the two treatments. The data clearly indicate that both the treatments induce the partial filling of the smallest pores with inducing the sharpening of the pore volume distribution and, as mentioned above, the decrease of the total pore volume. 4.4. Microtomography analysis

Fig. 5. SEM micrograph of a microsample of the Iron Age bone fragment after treatment 2 (pre-treatment with Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M). The presence of needle-shaped crystals.

• the inflection point at which capillary condensation occur does not shift considerably after consolidation, suggesting that the formed particles do not produce mesopore blocking. Nonetheless, the hysteresis loop became narrower after consolidation due to a possible reduction of the pore capillary forces, and the volume of adsorbed gas at a relative pressure of Ps /P0 = 0.9985 decreases suggesting the formation of a homogeneous coating in the pores. Concerning the hysteresis, all samples retain a type H3 loop: 47 loops of this type are given by non-rigid aggregates of plate-like particles but also if the pore network consists of macropores which are not completely filled with pore condensate [48]. As expected, this kind of loop reflect a non-uniform size/shape of pores, suggesting the presence of split-like pores. The calculations for the surface area were performed with the BET method. From BET adsorption-desorption isotherms we obtained surface area and pore volume of the analyzed samples. As reported in Table 1, we noticed that both the consolidation treatments affect the overall porosity of the consolidated bones. Regarding the consolidation 1, we observed a decrease in the value of the specific surface area of the bone and of the total pore volume of 54.9% and 34.8% respectively, while for consolidation 2 the decrease of the specific surface area and of the total pore volume was 53.2% and 31.3% respectively.

The consolidation effects induced by consolidation 1 and 2 were also investigated by means of microtomography, a radiographic imaging technique, able to achieve a spatial resolution close to 1 ␮m3 . The pseudo color images reported in Fig. 7, show how the matrixes of the untreated and of the treated bones interact with the X-ray source in terms of attenuation coefficient, which is related to mass density and elemental composition. The 3D images show the progressively increase of mineral density in both the consolidated samples, with particular attention to the decrease of the less dense region (in blue) in favor of a homogeneous denser phase (in green and red, Fig. 7). Fig. 7 shows that the consolidation effect is more homogeneous for consolidation 1 (Fig. 7 on the right) probably thanks to the higher penetration of the DAP solution into the porous matrix of the bone. For consolidation 2, the presence of Ca(OH)2 NPs probably induces the occlusion of part of the pores, reducing the penetration of DAP solution in the bulk of the bone and thus the precipitation of new HAP. This could explain the presence of a more homogeneous, green surface with respect the one subject to consolidation 2. This effect is also confirmed by SEM analysis. 4.5. SEM analysis Fig. 8 shows SEM micrographs of the Iron Age bone samples before (Fig. 5A) and after the two consolidation treatments (Fig. 5B for bone subject to consolidation 1 with a aqueous solution of DAP 1 M and Fig. 5C for bone subject to treatment 2, pre-consolidation with Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M). Fig. 8A indicates that before the consolidation the surface of the bone is inhomogeneous and characterized by the presence of

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Fig. 6. A. BET adsorption-desorption isotherms obtained for an Iron Age bone fragment before (filled triangles) and after (open circles) consolidation 1 (aqueous solution of DAP 1 M). B. BET adsorption-desorption isotherms obtained for an Iron Age bone fragment before (filled black circles) and after (open circles) consolidation 2 (pre-consolidation with Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M). C. Pore diameter distribution plots for an Iron Age bone fragment before (filled black triangles) and after (open triangles) treatment 1. D. Pore diameter distribution plots for an Iron Age bone fragment before (filled black circles) and after (open circles) consolidation 2. All the measurements have been carried out 2 weeks after the end of the application of the consolidant.

cavities. Both the consolidation treatments lead to the progressive recovery of a much more compact structure and to the formation of a homogeneous texture (Fig. 8B and C) with an apparent decrease of the dimensions of the open pores. This effect is more pronounced especially for bone sample subject to treatment 2 (Fig. 8C). Moreover, the SEM micrograph reported in Fig. 9, shows that the bone consolidated by means of consolidation 1 is characterized by the presence on the analyzed surface, of both crystalline and amorphous phases, indicated by white squares. In order to obtain information about the chemical nature of this phase, many spot sizes elemental analysis were carried out on different points of the treated bone (indicated by the white squares in Fig. 9A). EDX analysis show that the ratio between Ca:P in the bone subject to consolidation 1 was < 1 (Table 2), indicating the presence of calcium deficient calcium phosphate phases, while for treatment 2 the ratio between Ca:P was 1.67 everywhere, suggesting the presence of stochiometric HAP [15]. The mechanical properties of the treated samples have been investigated through Vickers Microhardness tests. Tensile stress test, that could be useful do obtain further information on the effect

of the consolidation treatments on the mechanical properties of the bone remains, were not possible in this case due to the too small dimension of the samples. Results show that we obtained an increase of the hardness of 42% and 56% for consolidation treatments 1 and 2 respectively. We can ascribe this difference to the formation of aragonite in presence of Ca(OH)2 nanoparticles, as confirmed by FTIR data. 4.6. Genetic analysis Regarding the genetic characterization of the three individuals from Kierzkowo, we were able to reconstruct the complete mitochondrial genome at medium-high coverage from each analyzed bone fragment both treated and untreated (Table S1). No significant human mitochondrial genome sequences were present in negative controls. The full sequencing results are reported in Table 3. Total number of raw and merged reads (DNA sequences) generated by the sequencing, is highly affected by the experimental bias due to the multiplexed capture experiments and PCR amplification cycles. Moreover, these values directly correlate with the total number of

Table 1 Specific area values and pore volume changes before (NT) and after consolidation 1 and consolidation 2 on bone remains.

NT Consolidation 1 Consolidation 2

Specific surface area (m2 /g)

Specific pore volume (ml/g)

Decrease of specific surface area (%)

Decrease of specific pore volume (%)

44.47 20.04 20.81

0.1344 0.0876 0.0924

54.9 53.2

34.8 31.3

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Fig. 7. 3D reconstruction of two fragments of an Iron Age bone samples before (NT), after consolidation 1 (aqueous solution of DAP 1 M, left side) and after consolidation 2 (pre-consolidation with Ca(OH)2 NPs in 2-propanol + aqueous solution of DAP 1 M, right side) on the right. The images report both sides of the analyzed samples. As clearly shown in the images, the blue, less dense, regions decrease after treatments in favor of a homogeneous denser phase.

Fig. 8. SEM micrographs of Iron Age bone fragments. Image A has been collected from a sample before the consolidation treatment; micrograph B was collected 15 days after the application of the consolidation treatment 1 (immersion in an aqueous solution of DAP 1 M until saturation); micrograph C was collected after the consolidation treatment 2: first soaking in 0.5 g/L Ca(OH)2 NPs dispersion in 2-propanol for 2 hours, followed by a completely drying under hood for one week, then a second soaking was done in an aqueous solution of DAP 1 M for 2 hours; the following two weeks the sample was maintained in a confined environment at RH = 75%.

reads mapping on the human mitochondrial reference genome. For these reasons, to better investigate the impact of the two consolidation treatments (consolidation 1 and consolidation 2) on the yield of paleogenetic analysis, we focused the comparison only on three main parameters (Table 3): • the percentage of reads mapping on the human mitochondrial reference genome, hereafter “percentage of endogenous DNA”;

• the average numbers of unique reads covering each position of the mitochondrial genome, hereafter “mean coverage”; • the percentage of mitochondrial genome positions covered by at least 5 different reads, hereafter “fold coverage”. Additionally, to investigate more in deep if the application of consolidants could be vehicle of modern human DNA contamination in the treated bones, we explored some molecular features of

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Fig. 9. A. SEM micrograph of Iron Age bone fragments after consolidation 1 (aqueous solution DAP 1 M). White squares indicate the points where the spot EDX analysis has been carried out. B. EDX spectrum collected in correspondence of the white square indicated by the arrow. The other spectra have a pattern similar to the one reported in B. Table 2 Average values of the atomic % obtained by the EDX analysis carried out on the points indicated by the black squares in Fig. 9A. Element

Atomic %

O C N P Ca

59.6 ± 1.7 10.3 ± 0.5 12.7 ± 0.5 10.3 ± 0.5 7.1 ± 0.5

the sequenced molecules (namely, percentage of damaged cytosine residues at both 3 and 5 ends, and average length) and estimating the proportion of authentic reads vs. putative contaminated modern human DNA sequences (contamMix) (Table 2). Compared to the untreated bones, we observed a decrease in the percentage of endogenous DNA in some consolidated fragments (K5.3 consolidation 1, K6.2 consolidation 1, K6.2 consolidation 2), while in the remaining fragments the values are similar or higher (K5.3 consolidation 2, K3.4 consolidation 1, K3.4 consolidation 2). In

two out of three individuals (K5.3 and K6.2), the mean coverage was lower in consolidated fragments, while in specimen K3.4 the coverage increases after consolidation. Despite the observed decreasing in some samples, mean coverage is relevant in all the analyzed fragments (between 362.91X and 11.72X), as confirmed also by the values of fold coverage showing that almost all the positions of the mitochondrial genomes reconstructed from the different bone fragments are covered by at least 5 different reads. No significant variations were observed in both average fragment length and proportion of damaged terminal cytosine, with values in the range of authentic degraded ancient DNA in all the analyzed bone fragments [49,50]. When comparing the results of the ContamMix test, we observed a decrease in the percentage of authentic reads in consolidated bones with respect to untreated fragments in two individuals (K5.3 and K6.2). This decreasing was not observed in the individuals K3.4 that, on the contrary, exhibit higher proportion of authentic reads in consolidated fragments than in untreated bone. Particularly in sample K6.2 consolidation 1 a relevant amount of sequenced DNA molecules could be derived by modern contaminants. Despite

Table 3 Results of the mitochondrial genome sequencing performed on the bones from the Neolithic site of Kierzkowo. Sample Name

Endogenous DNA (%)

Mean Coverage Fold Coverage (X) (≥5X) (%)

DMG 1st Base 3

DMG 1st Base 5

Average fragment length (bp)

ContamMix

Haplogroup

K3.4 consolidation 1 K3.4 consolidation 2 K3.4 untreat

18.56 12.52 6.18

126.52 39.05 14.79

99.95 99.69 94.27

0.32 0.30 0.31

0.33 0.31 0.31

58.09 61.54 54.69

0.98 0.98 0.92

U5b2b1a1 U5b2b1a1 U5b2b1a1

K5.3 consolidation 1 K5.3 consolidation 12 K5.3 untreat

0.08 5.54 5.35

14.94 14.84 31.76

97.51 97.95 99.78

0.32 0.27 0.33

0.38 0.29 0.30

51.48 63.11 56.74

0.98 0.91 0.99

U5b1d1a U5b1d1a U5b1d1a

K6.2 consolidation 1 K6.2 consolidation 2 K6.2 untreat

3.44 27.53 32.58

11.72 60.74 362.91

95.89 99.38 99.91

0.18 0.22 0.22

0.17 0.22 0.22

56.73 60.1 60.17

0.82 0.93 0.97

J1c J1c J1c

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the presence of some levels of contaminant molecules, for each individual the mitochondrial genome sequence reconstructed from the consolidated bone fragments belongs to the same haplogroup determined in the untreated bone. Even if additional tests will be necessary to limit experimental bias, the preliminary tests performed on the three bones from Kierzkowo showed that relevant amounts of authentic endogenous mitochondrial DNA molecules could be recovered from the bones after consolidation by means of consolidation 1 and consolidation 2, and mitochondrial genome sequence can be reconstructed with confidence in both untreated and consolidated bones.

5. Conclusions In this study, the results of the application of an innovative protocol for the consolidation of bone remains of historical and archaeological interest, based on the use of inorganic materials highly compatible with the bone matrix, are presented. Since bone remains found in archaeological sites usually need to be consolidated in order to avoid further rapid deterioration causing an increase of bone porosity and a decrease of both its density and hardness, our aim was to restore a continuous crystalline biocompatible network in the damaged regions, in order to preserve the remains and maintain long-term access to the biological information recorded in the bones. The efficacy in consolidating bones was evaluated by comparing results obtained with different techniques such as microtomography, nitrogen porosimetry, Vickers microhardness test, and Scanning Electron Microscopy. Through Vickers microhardness test and microtomography we noticed an increase in hardness (up to 56%) and mineral density respectively, thanks to the formation of new mineral phases, confirmed also by XRD and SEM. Furthermore, significantly reduction of pores volume and surface area was assessed by nitrogen porosimetry, confirming that the consolidation is not only limited to the surface but extend also in the inner part of the sample, thanks to the application method. After the consolidation treatment the bone matrix appears more compact and homogeneous. Furthermore, the data indicate that the formation of the new hydroxyapatite (induced by the application of a solution of diammonium phosphate) and of the aragonite (formed through the carbonation of Ca(OH)2 nanoparticles when the consolidation treatment has been carried out by applying them in addition to diammonium phosphate) occurred not only on the surface of the bone but also inside the porous structure, both reducing porosity values and conferring hardness to the porous bone network. The consolidation procedures presented in this paper, in principle, can be tailored for the consolidation of bones in different conservation status. For example, when the porosity of a bone is low, but it even needs to be consolidated, the consolidation treatment 1 based on the use of an aqueous solution of DAP can warrantee a good penetration of the consolidant into the porous matric of the bone. However, when the bone is strongly affected by decohesion problems and it is characterized by a high porosity, the consolidation performance of the DAP solution can be enhanced by the Ca(OH)2 nanoparticles that, through a carbonation reaction, promotes the formation of aragonite. Moreover, one of the most important innovation introduced in this work is that, for the first time, the efficacy of the consolidation treatment has been evaluated not only by considering the effect of the consolidants on the physico-chemical point of view, but also analyzing the impact of the consolidating materials applied onto the bone matrix on the molecular characterization of genetic material. Indeed, paleogenetic analysis on treated and untreated fragments from the same bone specimens demonstrate that the consolidating materials proposed in this study do not substantially

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affect the ability to recover endogenous DNA molecules. No change in damage patterns of the genetic material — due to the antiquity of the sample — seems to be induced by the consolidant protocols tested. Nevertheless, particular attention in consolidant application procedures and working environment should be considered to avoid contamination with modern DNA. The future research work could be mainly focused on the evaluation of the effects of consolidation materials and application methods on a long-term scale in order to test the long-term stability of the treated bone. Particular attention will be paid to the entity of the impact that the consolidation treatments have after some years on the recovery of the endogenous DNA molecules: the genetic analysis will be repeated one year or later on the same consolidated samples, in order to verify again the possibility to access to the DNA still present into the bone remains. Moreover, consolidation and DNA recovery tests will also be carried out on more diverse archaeological samples and with different molecular approaches (capture and shotgun) in order to retrieve both mitochondrial and nuclear DNA. Further analysis should be focused on testing the nondetrimental effect of consolidation on other molecular procedures like isotopic analysis and 14C dating. These results that are focused mainly on the consolidation of bone remains of archaeological interest, can be potentially useful also for the consolidation of paleontological remains, such as teeth, shell, and calcified tissues. This methodological and analytical approach followed may be useful to study the bone mineral chemistry and reactivity in vivo, enhancing our understanding of processes involved in healthy and pathological bone formation, shedding light on therapeutic approaches to osteoporosis in the elderly. Acknowledgments We thank Alicja Budnik of the Department of Human Biology, Cardinal Stefan Wyszynski University, Warsaw, Poland, for facilitating access to the bone samples from Kierzkowo and providing archeological information. This work has been funded by University of Florence (Nanoforbones project granted in the frame of the “Bando di Ateneo per il finanziamento di progetti competitivi per Ricercatori a Tempo Determinato” of the University of Florence Year 2016). Authors also thank the CSGI staff for the gas porosimetry measurements. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.culher.2019.07.022. References [1] R.E. Green, J. Krause, A.W. Biggs, T. Maricic, U. Stenzel, M. Kircher, A draft sequence of the Neandertal genome, Science 328 (5979) (2010) 710–722, http://dx.doi.org/10.1126/science.1188021. [2] K.I. Bos, V.J. Schuenemann, G.B. Golding, H.A. Burbano, N. Waglechner, B.K. Coombes, J.B. McPhee, S.N. DeWitte, M. Meyer, S. Schmedes, J. Wood, D.J.D. Earn, D.A. Herring, P. Bauer, H.N. Poinar, J. Krause, A draft genome of Yersinia pestis from victims of the Black Death, Nature 478 (7370) (2011) 506–510, http://dx.doi.org/10.1038/nature10549. [3] C.E.G. Amorim, S. Vai, C. Posth, A. Modi, I. Koncz, S. Hakenbeck, M.C. La Rocca, B. Mende, D. Bodo, W. Pohl, L.P. Baricco, E. Bedini, P. Francalacci, C. Giostra, T. Vida, D. Winger, U. von Freeden, S. ghirotto, M. Lari, G. Barbujani, J. Krause, D. Caramelli, P.J. Geray, K.R. Veeramah, Understanding 6th-century barbarian social organization and migration through paleogenomics, Nat. Commun. 9 (1) (2018), http://dx.doi.org/10.1038/s41467-018-06024-4 [art. no. 3547]. [4] A. Münster, C. Knipper, V.M. Oelze, N. Nicklisch, M. Stecher, B. Schlenker, R. Ganslmeier, M. Fragata, S. Friederich, V. Dresely, V. Hubensack, G. Brandt, H.J. Döhle, W. Vach, R. Schwarz, C. Metzner-Nebelsick, H. Meller, K.W. Alt, 4000 years of human dietary evolution in central Germany, from the first farmers to the first elites, PLoS One 13 (3) (2018) 0194862, http://dx.doi.org/10.1371/journal.pone.0194862.

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Please cite this article in press as: A. Salvatore, et al., Evaluation of Diammonium hydrogen phosphate and Ca(OH)2 nanoparticles for consolidation of ancient bones, Journal of Cultural Heritage (2019), https://doi.org/10.1016/j.culher.2019.07.022