In the field of cultural heritage conservation, bones remains from historical, archaeological and paleontological contexts are peculiar and precious finds. Particularly, human skeletal remains represent an enormous source of information about ancient humans in terms of their evolutionary and adaptation mechanisms, migratory flows and lifestyle (social and cultural behaviours, diet and diseases) [1, 2, 3, 4, 5, 6, 7]. Similarly, animal skeletal remains can provide information on past environments and on the social and economic organization of the populations which they are associated with [8, 9, 10, 11].
The chemical composition of bones is characterized by packed collagen fibers interconnected with a network of microcrystals of apatite with general formula Ca10(PO4)X2, where X usually indicates a hydroxyl group (hydroxyapatite, HAP). While the stoichiometric hydroxyapatite shows a Ca:P ratio of 1.67, the value typically observed in the organisms is widely variable due to several ion substitutions present in the hydroxyapatite of biological origin .
Ancient bone remains are typically discovered in critical conservation conditions, mostly in direct contact with the soil. The physico-chemical characteristics of the external environment (pH of the soil, moisture, temperature, environmental redox potential), the prolonged interaction with micro-and macro-organisms, together with the intrinsic physico-chemical properties of the remains, such as porosity and crystallinity, can strongly affect the conservation of the bones due to the deterioration of both collagen and mineral phase [13, 14, 15]. Consequently, in view of future studies, analyses and exhibitions, the recovery and handling of these objects are often critical.
Since the beginning of the twentieth century, the products typically used for consolidation treatments and fixing of the fragments are realized by natural and synthetic organic polymers (i.e. vinyl and acrylic polymers and copolymers) . Unfortunately, their scarce physico-chemical compatibility with the substrate and their low chemical stability, which results in possible depolymerization and cross-linking reactions, can lead to a rapid decrease of their performance, cause alterations of the bone matrix (yellowing and strong change of the porosity) and induce mechanical stresses due to the shrinking of these polymeric materials upon ageing. In addition, their degradation can seriously compromise their removal and the re-treatability of the object, besides hampering the analysis of biomolecules such as DNA and protein [16, 17]. Moreover, the application of organic polymers may significantly alter radiocarbon dating analysis by introducing external carbon atoms, especially in the case of poor information about the previous treatments or severe degradation processes occurred to those polymers.
In the last few years, inorganic nanomaterials have been proposed as a useful alternative to organic polymers [18, 19] for the conservation of different classes of works of art (i.e. consolidation of wall paintings and carbonatic stones, and deacidification of cellulosic materials such as canvas and papers). This approach allows obtaining consolidants that are physico-chemically compatible with the supports, highly stable and durable, and that can easily penetrate into the bulk of the porous materials. In particular, Ca(OH)2 nanoparticles dispersed in short chains alcohols have been successfully used as inorganic consolidants for carbonatic supports, ensuring an effective recovery of the mechanical properties of the powered matrixes [20, 21]. Some years ago, Ca(OH)2 nanoparticles dispersed in 2-propanol have been used also for the consolidation of archaeological bones to promote, through carbonation induced by the atmospheric CO2, the formation of aragonite, a metastable polymorph of CaCO3 characterized by strong mechanical properties . Nevertheless, according to the compatibility criterion, aragonite is not the best conservation material since bones are mainly composed by HAP. Therefore, in order to follow the compatibility criterion, the use of HAP for consolidation purposes on archaeological bones was evaluated in more recent studies. Indeed, HAP is typically employed for various biomedical applications, e.g. for bone regeneration and teeth reconstruction [23, 24, 25], and has recently given promising results for restoring the mechanical integrity of degraded stones [26, 27, 28]. Some studies have examined the possibility to induce the precipitation in situ of HAP: the first approach considered the reaction between a solution of a phosphate precursor, such as diammonium hydrogen phosphate (DAP), with the calcium present in the bone . Compared to typical organic treatments, like Paraloid® B-72 and Acrysol™ WS-24, the consolidation through DAP occurs without introducing incompatible compounds which significantly alter the surface morphology, the physiological porosity and the wettability. Unfortunately, the magnesium cation that is naturally present inside the bones in the form of binary salts, strongly affects the HAP crystallization processes, inhibiting the formation of a crystalline network of HAP .
In other studies [31, 32], the possibility to induce the in-situ growth of HAP was evaluated by immersing the bone fragments in an aqueous solution of DAP and a dispersion of Ca(OH)2 nanoparticles in alcohol. After this treatment, the bone matrix appears more compact and homogeneous thanks to the formation of new HAP and aragonite, both reducing the porosity and improving the mechanical properties of the bone network. Moreover, the examined consolidants do not compromise the results of the palaeogenetic analysis, allowing the retrieval of the complete mitochondrial genome of the ancient individuals without substantial impact on the quality of the data . Despite the promising results obtained in these studies, it would be necessary to develop an even more compatible and easily applicable consolidation treatment to be used in situ (i.e. directly at the moment of the excavation on fragile archaeological bones) and also on huge skeletal remains that cannot be treated by immersion. Considering these requirements, we aimed at the development of an innovative and highly compatible consolidation treatment based on the use of nanotechnologies. We also adopted a multi-disciplinary approach to evaluate its efficacy and impact on the bone matrix, especially in relationship to 14C dating and palaeogenetic analysis. The most important novelty proposed in the present study is the use of a dispersion of previously prepared HAP nanoparticles in combination with DAP and Ca(OH)2 nanoparticles. The HAP nanomaterial matches the compatibility criterion and, by synthesizing nanoparticles of proper dimensions, they have the capacity to penetrate the porous support and to fill in almost all fractures and cracks. The importance of HAP in the biomedical and industrial field has led to extensive research in different kinds of HAP synthesis methods [33, 34], but the most convenient and low-cost one is the precipitation process from homogeneous phase. In this work, we propose a fast, easy and cost-effective new method based on the precipitation of HAP nanoparticles from an aqueous solution of precursors (calcium nitrate, Ca(NO3)2∙4H2O, and DAP) adjusting synthesis parameters to minimize both particles size and their aggregation. Moreover, the idea at the basis of the proposed methodology was to try fixing the obtained HAP nanoparticles to the substrate by exploiting the binding properties of a mixture containing DAP and Ca(OH)2 nanoparticles. Indeed, the in situ precipitation of calcium phosphate from this mixture has already been demonstrated effective : according to this new approach, the precipitation of calcium phosphate where HAP nanoparticles were previously deposed was expected to act as a binder in a mortar.
Another important novelty of this work was the multidisciplinary approach adopted to evaluate not only the performance of the proposed consolidating treatment but also its possible drawbacks for 14C dating and palaeogenetic analyses. The efficacy of the consolidation was examined in terms of the impact of the treatment on the physico-chemical and mechanical properties of the treated bones. In particular, their morphology – homogeneity and surface cohesion -, porosity and micro-hardness were assessed. To investigate the impact of the consolidation treatment on the retrieval of endogenous DNA, we applied biomolecular technologies typically used in the field of ancient DNA (aDNA) to recover the mitochondrial genome (mtDNA) from both treated and untreated fragments deriving from the same bone sample. The consensus sequence was reconstructed and assigned to the respective mitochondrial haplogroup; after checking the authenticity of the data, the results obtained from the consolidated and the untreated fragments were compared to spot any significant impact on the quality and reliability of the genetic data. As far as radiocarbon dating is concerned, tests were carried out to determine whether the consolidation treatment might introduce contamination that could not be eliminated by applying the typical procedures used to extract collagen and lately purify it from natural exogenous substances : to this purpose, both untreated and treated bone samples were dated.