Hydroxyapatite scaffolds derived from deer antler: Structure dependence on processing temperature

Highlights

• Hexagonal HA enriched in Mg & Na (as in human bone) was obtained from cortical & trabecular sections.

• Calcination at 950 °C ensured organic material removal and re-crystallization in both sections.

• Re-crystallization was proved by CaO peaks presence (XRD) and carbonated HA peaks absence (EELS).

• Earlier calcination in trabecular section with higher mass loss & larger crystals than cortical

• Morphological differences as 52% porosity in trabecular (pores >200 μm); 4% in cortical (<10 μm)

Abstract

Discarded antlers from deer are proposed as a promising alternative CaP-based bone graft to fulfil specific unsolved clinical requirements, such as osteoinductive properties or an optimal balance in stability/resorbability. Moreover, depending on the location of the bone defect and the type of bone lost (cortical versus cancellous), adequate morphological/mechanical properties for indicated biomaterials are needed. At the present work a detailed study of the physicochemical properties of two bioceramics obtained from the cortical and the trabecular sections of deer antler is presented. The influence of temperature on both bioceramics was also evaluated in depth to guarantee removal of organic material, analyze the compositional changes for high temperatures (up to 1100 °C) and study how their specific morphological features can influence these modifications. Morphological evaluation (SEM, porosity) of both final bioapatites (cortical and trabecular) was assessed, together with composition (ICP-OES, EDS, FT-Raman, XRD, TEM) and mechanical properties (nano-indentation). Optimal temperature for calcination was selected, through a thermogravimetric analysis, to ensure: the removal of organic material and a re-crystallization process (carbonate group decomposition) in both sections. Main contribution of hydroxyapatite (Ca₅(PO₄)₃(OH)) in hexagonal phase was found, structure similar to human bone, with the presence of periclase (MgO). A Ca/P ratio in the same range as porcine and bovine bones, and with trace elements, such as Mg and Na, that play relevant roles in osteogenic metabolism was also detected. The dense and compact structure in cortical section and the spongy-like structure in the trabecular one, with pores >200 μm in diameter occupying a surface of 52 ± 8% were characterized. Related to these morphological properties, the same calcination temperature was proven to yield larger crystals in the trabecular section, given the higher availability of space for the crystal to grow (lower density of material).

Introduction

In recent years, hydroxyapatite (HA)-based graft materials of biological origin as bone substitutes have attracted greater interest than those of synthetic origin, given that their behavior is biologically more active compared with synthetic equivalents [[1], [2], [3], [4], [5]]. In contrast to commercial synthetic HA, the biological version is enriched with essential elements (such as Mg²⁺, K⁺, Na⁺, etc.) that have specific roles of relevance in bone defect healing, and it has lower crystallinity with disordered nanostructures providing unique chemical and physical properties [2,3,6]. Therefore, when the nanoscale properties and composition are preserved, a better performance of biological HA is expected [1,6,7].

Most sources of biological CaP currently available to clinicians for the repair of critical bone defects are in the form of xenografts (from bovine or porcine bones), allografts (from other human donors) or autogenic bones (patient's own bones). While all have been used as standard biomaterials for many years, they present some disadvantages, such as low bone fusion rate and disease transmission risks observed in allogenic grafts [8]. In the case of autogenic bones, poor functionality caused by overly rapid resorption levels has been observed together with pain at the site of bone harvesting [9,10]. As for commercial xenogenic bone substitutes, these are widely available and present reasonable efficacy in repairing bone defects [11], but they imply the sacrifice of the animal; in addition, it is worth mentioning their inability to reach sufficient height and width in medium-sized bone defects and their potential to cause immune response in the patient [12,13].

New approaches based on renewable resources are being investigated, seeking to guarantee sustainability. One such approach is exploring the potential of the marine environment, especially discards from the fishing industry. Several authors have proposed different alternatives, such as fish bones from tuna or sword fish [1], which have a composition based on calcium orthophosphates. This waste from the canning industry is produced by the biggest fisheries in the world. An alternative comes from shark teeth [14,15], which are a fishery discard mainly composed of fluorinated hydroxyapatite. All these bioapatites are subjected to heat treatments at high temperatures (>1000 °C) to remove organic material. At these temperatures the absence of proteins in the final product is guaranteed, as all known proteins are ashed at these temperatures [16], avoiding or minimizing the risk of disease transmission or immune responses to extremely low level. Other possibilities are the shells of crustaceans, such as crabs [17], another waste from the food industry, these shells are essentially composed of calcium carbonate and require a hydrothermal exchange process to obtain hydroxyapatite. The bones from Cephalopoda, such as cuttlefish (Sepia officinalis), subjected to a hydrothermal transformation of aragonite can also be used [18].

Apart from the marine environment, another sustainable source of CaP- based bone grafts is the discarded antlers from deer (Cervidae spp), given their apatitic structure that is similar in composition and morphology to certain human and animal bones [19]. In fact, cervids are the only animals that have antlers that are made of true bone, since that apart from the mineral structure, collagen, non-collagenous proteins and water are also present [[20], [21], [22], [23], [24]]. The advantages of this source begin with its formation: antler growth occurs during the first four months [25] starting to grow in spring, calcifying in autumn, to be finally discarded by the animal in late winter [26,27]. This cycle guarantees high annual availability. Moreover, using deer antlers has the double advantage of not requiring animal sacrifice and of promoting the re-use of waste and discards, which respectively support ethics and animal welfare, as well as sustainability regulations. In relation to mechanical performance, one of the most important intrinsic parameters is Young's modulus of elasticity (a measure of stiffness) which in antler is in the ranges between 5 and 7 GPa depending on whether it comes from captive or free-ranging deer [22]. Moreover, a value of 7.30 ± 0.30 GPa was measured by Currey et al. [20] for the entire antler in wet conditions, to simulate the internal bone environment. In comparison, Young's modulus for human bone was measured by Rho et al. [28], with values in the range 10–15 GPa for trabecular bone, depending on ultrasonic or mechanical measurements.

Zhang et al. [19] hypothesized that, given its similarities to human bone and the previously mentioned advantages, calcinated antler cancellous bone (abbreviated by them as CACB) may serve as an ethically acceptable xenograft substitute for bone defect repair. These authors demonstrated the efficacy of CACB for bone regeneration inducing neovascularization and osteogenesis within mandible defects at levels comparable to other grafts already on the market. Later, Zhang et al. [29] proved the compatibility of CACB scaffolds with loaded growth factors, as well as their effects on the osteogenic potential of these composites in vitro and in vivo. The most marked positive effects on rBMSC cell attachment, proliferation, osteogenic differentiation and mineralization were found for icariin. Meng et al. [30] concluded that the effect of deer age should be considered when optimizing the physicochemical and biological properties of CACB for bone defect repair.

It is well known that depending on the location of the bone defect and the type of bone lost (cortical versus cancellous), the adequate properties for indicated biomaterials may vary considerably. Porosity, topography and mechanical strength should always be balanced with the mechanical needs of the particular tissue that is going to be replaced. Bone grafts mainly composed of cortical bone will present superior structural strength with a Young's modulus in MPa thousand times greater than cancellous bone grafts. Cancellous or trabecular grafts, on the other hand, have the ability to induce early and rapid new bone formation [31]. The clinician will select the most appropriate bone graft based on the demands of the pathology or surgery [32].

Deer antler is described as being composed of four different histological zones from the periphery to the center of the antler: the first consists of osteoid located just below the velvet; then a zone of osteonic bone composed of lamellar compact bone; the third is a transition region between the osteonic bone and the trabecular bone; and the fourth is a central zone consisting of trabecular bone [21,33]. No research studies to date have evaluated the compact (cortical) section and the trabecular section as two differentiated bioceramics. A detailed study of the physicochemical properties of bioceramics obtained from the cortical and trabecular section of deer antler is presented in this work. The influence of temperature on both bioceramics was also evaluated in depth to analyze compositional changes for high temperatures (up to 1100 °C) and study how their specific morphological features (such as porosity level) can influence these modifications.