Functionalization of Ceramic Coatings for Enhancing Integration in Osteoporotic Bone: A Systematic Review

: Background: The success of reconstructive orthopaedic surgery strongly depends on the mechanical and biological integration between the prosthesis and the host bone tissue. Progressive population ageing with increased frequency of altered bone metabolism conditions requires new strategies for ensuring an early implant ﬁxation and long-term stability. Ceramic materials and ceramic-based coatings, owing to the release of calcium phosphate and to the precipitation of a biological apatite at the bone-implant interface, are able to promote a strong bonding between the host bone and the implant. Methods: The aim of the present systematic review is the analysis of the existing literature on the functionalization strategies for improving the implant osteointegration in osteoporotic bone and their relative translation into the clinical practice. The review process, conducted on two electronic databases, identiﬁed 47 eligible preclinical studies and 5 clinical trials. Results: Preclinical data analysis showed that functionalization with both organic and inorganic molecules usually improves osseointegration in the osteoporotic condition, assessed mainly in rodent models. Clinical studies, mainly retrospective, have tested no functionalization strategies. Registered trademarks materials have been investigated and there is lack of information about the micro- or nano- topography of ceramics. Conclusions: Ceramic materials / coatings functionalization obtained promising results in improving implant osseointegration even in osteoporotic conditions but preclinical evidence has not been fully translated to clinical applications. Analysis of the site of implant of functionalized materials described in the preclinical papers.


Introduction
In the twenty-first century, performing an implant procedure, ranging from screw insertion to total joint replacement, has become practically "routine" in orthopaedics and traumatology as well as in dentistry. Besides the surgical technique, other several factors are involved in determining the success of the implant, not just as a procedure but as a final outcome in term of osteointegration. Osteointegration is a biological concept that develops as a multistage process in which different cell/tissue events, reparative/regenerative mechanisms and local and systemic factors concur to the integration of a foreign body into the host bone [1,2].
The biomaterial surface properties, as surface energy, chemical composition, geometry, topography, wettability, roughness represent crucial players involved in the establishment of an optimal bone/implant interface and capable of influencing the type of interaction between biomaterials and biological environment. Over the years, the in-depth research and analysis of biomaterials properties has led to the understanding that functionalizing biomaterials can represent a winning strategy in determining the success of the osteointegration process [3]. To functionalize means to modify the biomaterial surface properties conferring new functionalities in order to achieve specific goals. Through the functionalization, the applicability of the original biomaterial is further broadened to serve specific purposes [4].
Physical, chemical and biological functionalizations represent the available strategies for tailoring a material's surface based on the technique or surface modifier taken into consideration. Physical functionalization is performed to improve the topographical properties of biomaterials, the pore size and the morphology in order to act onto cell differentiation, migration and adhesion. Chemical functionalization is pursued to modify composition, energy or surface charge to direct and influence cells interaction and adhesion. Finally, biological functionalization is performed with covalent or non-covalent immobilization or conjugation of biomolecules, drugs, growth factors and peptides, which can elicit a specific biological response directing cell behaviours or inhibiting adverse reactions [4,5].
A good quality of bone bed certainly represents a positive prognostic factor for the osteointegrative process; however, quite often orthopaedics are faced with patients whose bone status is compromised by systemic diseases or disorders, such as osteoporosis or diabetes, immunologic pathologies, oncologic diseases requiring anticancer therapies or various other age and lifestyle-related pathological conditions [6][7][8].
Osteoporosis (OP) is one of the most common musculoskeletal disorders in which low bone mass and bone micro-architecture and extra-cellular matrix (ECM) impairment leads to an increase of fracture susceptibility [9].
According to recent epidemiologic data, the growing prevalence of OP, also due to the increase in life expectancy, has led to a higher incidence of fragility fracture, experienced by 44% of women and 25% of men [10,11].
Although from a clinical perspective, OP is not considered a contraindication for implant placement [12], several experimental data report that OP may jeopardize bone healing and regeneration, thus reducing osteointegration and worsen the support provided by the host bone [13].
That is why many research studies focus on the development of smart biomaterials with improved features aimed at enhancing osteointegration and implant primary and biological stability. In very challenging situations of poor bone quality and quantity, such as in OP, ceramics, as bulk material or coating, seem to be the most promising choice to promote bone regeneration processes improving bone healing and osteointegration [14,15]. In fact, the results of a recently published review and meta-analysis of preclinical research on osteointegration in osteoporotic conditions highlighted that ceramics, and in particular calcium phosphate (CaP), improve one of the main histological osteointegration parameters (i.e., bone to implant contact-BIC), in comparison with uncoated implants [16]. It has been widely demonstrated that CaP materials are able to favour bone regeneration due to biological affinity with bone tissue: thanks to a dissolution process, the increasing Ca and P ion concentration in the local microenvironment significantly improves the adhesion of osteoblast-like cells and mesenchymal stem cells on implant surfaces. The subsequent precipitation of a biological apatite with a composition similar to bone serves as a matrix for cell attachment and growth. Concurrently, osteoclasts (OCs) are actively involved in the dissolution process of biological apatite, thus favouring new bone growth into fissures and defects left by the resorbing process in the post-operative period [17][18][19].
In light of these aspects, the attention has now been focused on the use of functionalized ceramic materials used both as synthetic bone graft substitutes than as a thin coating on metallic implants with the aim to further improve the biological properties of biomaterials thus favouring and improving fixation and osteointegration, especially in OP bone. The functionalization strategies performed both through the enrichment of the bulk ceramic material with trace ionic species or drugs as well as with biological entities as growth factors, drugs, peptides or cells, have emerged as a very promising alternative to induce cellular response and further stimulate endogenous reparative/regenerative mechanisms.
Therefore, the aim of this study is to systematically review the preclinical and clinical literature on the subject of bone osteointegration in osteoporotic conditions. Since the effect of ceramic materials on osteointegrative processes in osteoporosis has been already demonstrated [16], the present review aims to provide a view on the state of the art on the functionalization strategies of ceramic materials or coatings and their effects on the osteointegration process in osteoporotic conditions also assessing which of these approaches have been or can be translated to the clinical application.

Summary of the Literature Results
The a priori search retrieved 125 articles from www.pubmed.com and 208 from www.webofscience.com. After screening, several articles (242) were excluded: 51 studies did not evaluate ceramic materials or ceramic coatings; in 64 studies the osteoporotic or osteopenic condition was not present; 48 studies were in vitro; in 9 studies an ectopic implant was performed; 29 were reviews; 15 cadaveric studies; 3 studies were not related to the topic of the present review and in other 4 studies no analysis related to osteointegration has been performed. Finally, in 19 studies, no functionalizations of ceramic material or coating were performed. Therefore, a total of 91 papers were recognized as eligible for the review and after the use of a public reference manager (Mendeley 1. 19.3) to eliminate duplicate articles, 52 papers have been evaluated: 47 preclinical studies and 5 clinical studies ( Figure 1). The results of the research were reported stratifying the preclinical papers according to small, medium and large animal models and clinical studies.

Preclinical Studies
Preclinical studies were evaluated considering i) the animal model and the strategy adopted to induce OP or osteopenia; ii) type of surgery and site of implant; iii) the main characteristics of ceramic material/coating and the type of functionalization; iv) the experimental set-up (study groups, experimental times and analyses) and v) the main results obtained on osteointegration. Out of 47 preclinical studies, 31 were conducted in small animal models, 8 in medium sized animal models and 8 in large sized animal models. All studies were conducted in osteoporotic animal models.

Small Animal Models
Rodents are the most used preclinical model for the evaluation of implant coatings in OP. A single study employed a mouse model, while 30 rats were employed in the others. In all studies, OP was established via bilateral ovariectomy, in 4 studies also combined with low calcium diet. In the mouse study a genetic model was created. Only one study employed male rats. In the mouse study, a composite material of ceramic and bioglass, with and without brain-derived neurotrophic factor (BDNF), was implanted in femurs.
As for the rat model, 20/30 implant sites were femur, 6/30 were tibia, 3/30 were calvaria and one study was in the mandible. Among femoral implantation, in 8/20 studies, materials were placed in the medullary cavity. In 17/30 studies, the implanted material was ceramic, 12 were titanium (Ti) implants while in one study, both ceramics and different hydrogels compositions were used.

Summary of the Literature Results
The a priori search retrieved 125 articles from www.pubmed.com and 208 from www.webofscience. com. After screening, several articles (242) were excluded: 51 studies did not evaluate ceramic materials or ceramic coatings; in 64 studies the osteoporotic or osteopenic condition was not present; 48 studies were in vitro; in 9 studies an ectopic implant was performed; 29 were reviews; 15 cadaveric studies; 3 studies were not related to the topic of the present review and in other 4 studies no analysis related to osteointegration has been performed. Finally, in 19 studies, no functionalizations of ceramic material or coating were performed. Therefore, a total of 91 papers were recognized as eligible for the review and after the use of a public reference manager (Mendeley 1.19.3) to eliminate duplicate articles, 52 papers have been evaluated: 47 preclinical studies and 5 clinical studies ( Figure 1). The results of the research were reported stratifying the preclinical papers according to small, medium and large animal models and clinical studies.

Preclinical Studies
Preclinical studies were evaluated considering (i) the animal model and the strategy adopted to induce OP or osteopenia; (ii) type of surgery and site of implant; (iii) the main characteristics of ceramic material/coating and the type of functionalization; (iv) the experimental set-up (study groups, experimental times and analyses) and (v) the main results obtained on osteointegration. Out of 47 preclinical studies, 31 were conducted in small animal models, 8 in medium sized animal models and 8 in large sized animal models. All studies were conducted in osteoporotic animal models.

Small Animal Models
Rodents are the most used preclinical model for the evaluation of implant coatings in OP. A single study employed a mouse model, while 30 rats were employed in the others. In all studies, OP was established via bilateral ovariectomy, in 4 studies also combined with low calcium diet. In the mouse study a genetic model was created. Only one study employed male rats. In the mouse study, a composite material of ceramic and bioglass, with and without brain-derived neurotrophic factor (BDNF), was implanted in femurs.

Mouse Model
Kauschke et al. [21] created a genetic model of OP to investigate the effect on bone regeneration of α-tricalcium phosphate (α-TCP)-based hydroxyapatite (HA) containing bioactive CaP-silicon dioxide (SiO 2 ) glass particles with or without BDNF functionalization. The outcomes of implantation in the distal femoral metaphyseal gap highlighted a greater regenerative potential in the healthy group than in OP ones. The authors suggest that a higher concentration of BDNF should be needed in order to observe effects also in the osteoporotic condition.

Rat Model
Almost one half of the included articles (15/30) investigated the performance of Ti implants coated or combined with different materials/adjuvant in OP rats. Alghamadi et al. [22] investigated a CaP coating functionalized with alendronate (ALN), assessing that the simultaneous release of CaP and BPs act positively both on bone formation and on the rate of bone resorption in comparison to CaP or BPs single coatings also in OP. Pyo SW et al. also investigated CaP coatings with the addition of zoledronate (ZOL) at different concentration (from 0 to 800 µg/mL), immobilized on Ti screws [23]. In this case, at the highest ZOL concentration corresponded the best result in terms of bone growth in both healthy and OP, while BIC values were found lower in comparison to the control group. No data of ZOL release from CaP coating around the implants have been reported. Kettenberg et al. instead filled the holes of a metallic screw with a hyaluronan derivative hydrogel containing HA and ZOL. Their findings showed that the hydrogel acted as carriers but at the same was mineralized by the presence of HA; ZOL prevented hydrogel resorption, thus promoting bone regeneration [24].
The group by Li et al. examined in different researches the loading of traced elements, as strontium (Sr), magnesium (Mg) or zinc (Zn), onto HA coating of Ti materials implanted in femurs. The HA coating functionalization with Mg or Zn was proven to improve maximum push out force and BIC values, suggesting a better osteointegration in comparison with HA alone [25,26]; similar results were obtained with 10% SrHA coating, which proved to have a trophic effects on the trabecular architecture of bone around implants, increasing bone density and bone formation [27]. In light of this result, the effectiveness of the 10% SrHA coating was tested after treatment with subcutaneous injection of ZOL, to mimic clinical situations of patients undergoing systemic BPs therapy, showing good outcomes in terms of bone density and the main microtomographical trabecular parameters [28].
Tao et al. also adopted Sr coating to improve the osteointegration of Ti and ceramic materials in OP and osteopenic rats. The work group identified a direct correlation between the percentage of Sr ions used for coating (range tested: 5%-20%) and implant osseointegration measured by microtomography, histology and mechanical tests [29]. Although experimental coating with Mg and Zn showed good results in terms of implant integration and fixation and bone formation, the group treated with 10% Sr coating had significantly higher values of tested parameters [30]. Zhang et al. set up an analogue experimental model testing lower percentage of the same ions (2.5% of Mg, Zn and Sr) [31]. Even in these cases the addition of trace elements did not alter HA implant porosity and general morphology, while the best results in terms of bone density and BIC values were obtained in presence of Sr and Zn, in comparison to Mg or HA alone. Furthermore, the addition of Si, Zn and Sr to ceramic materials proved to be effective not only in improving implant osseointegration in osteoporotic environment but also in reducing bone loss and thus showing a potential therapeutic application for the pathology itself [32]. Tao et al. evaluated if the administration of drugs, in this case parathormone (PTH) injected 3 times/weeks subcutaneously at 60 mg/kg, could further improve the performance of 10% Sr-HA coated Ti implant in promoting bone formation. Results confirmed the hypothesis, showing higher bone formation in the group treated with PTH not only at the interface with implants but also in the medullary cavity in which implant was placed [33]. On this path, the same group tested Sr coating on calcium phosphate cement (CPC) paste and observed that the concurrent administration of BMP improved osteoinductive and osteoconductive processes, both histologically and biomechanically [34].
Moreover, for ceramic materials, the combination with Sr ions led to the most promising results. Baier et al. performing morphological analysis on the materials, observed a less homogenous material profile in Sr-containing calcium phosphate cement (CPC), which seemed to promote bone ingrowth locally around the implants but no systemic effects driven by Sr presence were evidenced with bone mineral density (BMD) evaluation [35]. Cardemil et al. highlighted that Sr/CaP granules and HA granules both induce quantitatively comparable healing of trabecular bone defects, driven by different mechanisms [36]. Histological and histomorphometrical evaluations showed different distribution of newly formed bone, linked to the different rate and timing of material resorption. Sr group exhibited higher expression of osteoblastic genes and reduced presence of osteoclastic activity in comparison with the control group [36].
Results obtained from the evaluation of osteogenic potential of Sr incorporated HA micro-granules are in line with these evidences. Chandran et al. demonstrated in a long-term OP-induced aged model that the combination of Sr and highly porous HA promotes complete bone healing, showing a regeneration efficacy higher than the HA group, also confirmed by microCT evaluation of the trabecular bone status [37].
Thorman et al., in a similar in vivo model, also used time-of-flight secondary ion mass spectrometry (TOF-SIMS) technology to detect ions released from Sr modified CaP cements. The matching between results from TOF-SIMS and evidence from histology and immunohistochemistry (IHC) allowed to attribute the highest bone formation at the interface between SrCPC cement with a high release of Sr ions from the material, which locally acts promoting bone growth [38].
SrCPC cements were compared with porous scaffold silica/collagen xerogel and monolithic silica/collagen xerogel in a study in which bone formation was assessed with F-18-sodium fluoride (NaF) dynamic PET-CT (dPET-CT). The evaluation of the movement of tracer into and out of the bound bone compartment, reflecting fluorapatite formation, showed the highest values in Sr containing materials, suggesting a greater bone formation in comparison to others and at the same time proposing a different method for studying the efficacy of materials for bone regeneration in OP [39].
Staying in the field of modification of ceramics materials, the group of van Houdt et al. evaluated the performance of CPC (30 wt %) enriched with PLGA microparticles (70 wt %). In one study, the degradation rate of CaP/PLGA materials was found to be higher and faster in comparison to Bio-Oss ® , which instead showed better ability to promote bone formation. The loading of CPC/PLGA (60-40 wt %) materials with ALN revealed to be successful in promoting bone formation in comparison to material without pharmaceuticals. The rate of degradation of CPC/PLGA in comparison to CPC alone allowed to control the release of ALN, maximizing the local effect [40].
ALN was also used to synthesize a multifunctional HA, in combination with Fe 3 O 4 , with magnetic properties, which proved to reduce osteoclast activity and promote bone formation via osteoblasts (OBs) activation [41]. The influence of PLGA for osseointegration in OP was also studied by Jeong et al. who combined it with β-tricalcium phosphate (β-TCP) screws, showing that such biocomposite screws increase microtomographical parameter values of bone formation in ovariectomized (OVX) groups [42]. As an alternative to the use of BPs, melatonin was loaded onto a calcium aluminate (CA) scaffold. Melatonin (Mel) proved to act on the overall bone metabolism, balancing bone formation and resorption, promoting bone healing. The addition of platelet rich plasma (PRP) did not exert any trophic effects, probably because of difficulties in selecting the proper administration times, which should consider the different mechanism of actions [43]. Also, doping lithium (Li) onto CPC proved to induce early endochondral ossification and new bone formation in comparison to CPC alone, preserving the characteristics of bioactivity and biocompatibility of ceramic materials [44]. While Fang et al. observed that the direct loading of simvastatin (SIM) on implants to substitute systemic administration alter the size of HA crystals, making them smaller and more irregular [45].
One of the most important aspects in bone healing is the onset of adequate angiogenic process. Wu et al. loading CPC cements with icariin, observed a strengthening of both osteogenic and angiogenic phenomena, evaluated with Microfil perfusion analysis, compared to CPC alone [46]. In addition, overall parameters of BMD, serum calcium levels and bone bending strength appeared ameliorated in OVX rats, indicating a systemic, as well as local effect. Xia et al. had the same results locally, evaluating the effect of Ca, Mg, Si containing akermanite bioceramics, with the best outcome in comparison to β-TCP alone [47].
Liu W. et al instead focus the attention on the influence that microenvironmental pH can exert on fracture healing and bone formation in osteoporosis, testing alkaline biodegradable implants. The dosage of this parameter after implantation of 10% Sr-substituted CA and β-TCP showed that the presence of the materials induced a rise in pH level, which correlate with higher bone formation [48].
Liu X. et al., in the only retrieved dental study, seeded rat BMSCs infected with human osteoprotegerin (OPG) adenoviruses in order to express the higher level of the OPG gene onto HA scaffold. Results showed that the construct was able to act on bone homeostasis, promoting new bone formation and regulating resorptive processes, putting itself up as a good candidate for the treatment of bone fracture/defects in condition of compromised bone status [49].
Hauser et al. tested L51P, a BMP-2 variant able to restore BMP2-mediated osteoblasts differentiation inhibiting BMP2 antagonist, in combination with β-TCP cylinders [50]. To maximize the potentiality of the materials in osteoporotic condition, ALN was subcutaneously injected. The results confirmed that the synergistic effects of BMP2 and L51P augmented bone formation after surgery but on the other hand the reduction in β-TCP degradation after ALN administration, opened up the question about the concurrence of chemical composition and osteoclasts activity in material resorption.

Medium Animal Model
The research retrieved 8 preclinical studies performed on rabbits. In 3/8 studies, OP was induced with bilateral ovariectomy surgery, while in other 3/8, a combination of ovariectomy and corticosteroid administration, mainly methylprednisolone sodium succinate, was used to induce the condition. In the last 2/8 studies, a temporary OP was induced with corticosteroid administration. To evaluate the regenerative potential of ceramic functionalization strategy, in 4/8 studies defects were realized and directly filled with ceramic materials combined with different organic or inorganic molecules, while in 3/8 studies, the functionalization served as a coating for metallic implant. One study investigated a combination of polymeric material screw-shaped and functionalization with Sr. Femur was used as implant site in 6/8 studies, tibia in 1/8 and mandible in 1/8 (Table A2 in Appendix A).
Moreover, in this animal model the use of bisphosphonates recurs. Gong et al. functionalized a calcium silicate powder (CPCS) with 0.5% risendronate (RA) obtaining an increased bone formation in the defects treated with RA-CPCS 0.5%, in comparison with the ceramic material alone and a preventive effect on bone resorption. In addition, the authors found a significantly up-regulation of several genes related to osteogenesis in comparison to CPCS group [51]. Zarins et al. investigated the regenerative potential of HA/TCP ceramic granules with or without 5% strontium observing no statistical difference of bone regeneration among operated and non-operated samples in OP animals. Nevertheless, Sr functionalization increased the expression of different factors related to osteocytes activity as OPG or nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) [54].
The use of inorganic molecules to impart new functionalities of ceramics is reported by Yu et al. which functionalized CPCS coatings with two different Zn contents for Ti 6 Al 4 V pin implanted in osteopenic rabbit. The authors observed a significant increase in new bone formation and microarchitectural bone parameters around the implant coated with the higher Zn content at early (1 month) and late experimental times (3 months) in comparison to the other groups of the study [55].
Among the functionalization performed with biomolecules, the existing papers reported a functionalization of a mesoporous silica with osteostatin or of HA with adiponectin or fibronectin. Lozano et al. adopted the first strategy investigating the recently characterized osteogenic effect of PTHrP epitope (107-111) named osteostatin. Even if no quantitative measurements have been performed in terms of bone regeneration, osteostatin functionalization induced an increased staining for key factors involved in bone regeneration process as proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2), osteopontin (OPN) and vascular endothelial growth factor (VEGF) [56].
Luo et al. and Plaza et al. respectively functionalized HA with adiponectin/matrigel (APN + matrigel) or fibronectin. In the first approach, the authors used matrigel to control the release hormone from HA surface and observed a weaker tartrate-resistant acid phosphatase (TRAP) staining in APN-matrigel-HA group in comparison with the other groups, as well as a significant new bone formation [57,58].
Plaza et al. used fibronectin functionalized HA and coat screw obtaining a significant improvement in trabecular bone microarchitecture parameters, even if not significant in comparison with the HA coating or control [58].

Large Animal Model
Eight studies on large animal models (7 sheep and 1 goat) were retrieved from the search strategy. In 6/8 studies OP was induced by bilateral ovariectomy while in 2/8 studies, an osteopenic condition was induced but no indications about the method to induce the condition has been provided. In addition, in 2/8 studies, ovariectomy surgery was also combined with low calcium diet while in other 2/8, a corticosteroid administration was associated with surgery. Finally, in 1/8 studies, ovariectomy surgery, low calcium diet and corticosteroid administration were combined.
The in vivo biological response in terms of osteointegration of functionalization strategies was evaluated realizing confined defects (6/8) in different anatomical regions: in 4/6 at vertebral level, 1/6 in the iliac crest (ALT) and 1/6 in the cortical bone of the thigh. In 2/8 studies, the functionalized ceramic materials were used to coat Ti device implanted in cancellous bone.
The types of functionalization evaluated in large animal models are mainly followed with the use of bioactive biomolecules (6/8). In 1/8 studies the synergistic functionalization of ceramics with organic and inorganic molecules was performed while one study (1/8) adopted BPs as drug to load ceramic material (Table A2 in Appendix A).
In the studies performed by Gunnella et al., the researchers evaluated the regenerative potential of PLGA fibre-reinforced brushite-forming cement (CPC) with BMP-2 or BB-1 at different dosages with augmented bone formation capability. In both of studies, CPC + fibres and/or CPC + fibers + BMP-2 or BB-1 significantly improved the main parameters related to bone formation, resorption and structure in comparison with control, with a dose dependent mechanism [59,60].
James et al. treated lumbar vertebral defects with hyaluronic acid (HyA) combined with HA/β-TCP further functionalized with rhNELL-1 protein, an osteoinductive factor with both pro-osteogenic and anti-osteoclastic properties, in comparison with HA-β-TCP at two different dosages (0.9 and 2.25 mg). An improved bone quality was detected by microtomographic investigations as BMD significant increase in a dose-dependent manner in defects treated with rhNELL-1 in comparison with control as well as BV/TV and Tb.N showed a significant increase at both dosages. The histomorphometric analysis further confirmed the results obtained with microCT, highlighting an improved cortical and cancellous bone regeneration [61].
A more traditional approach was pursued by Verron et al., who evaluated the performance of a calcium deficient apatite (CDA) functionalized with ALN. In this study, the authors highlighted an improvement in bone content and microarchitectural properties of bone in comparison with CDA alone, within 1.2 mm from the implant, thus suggesting an anabolic effect distance dependent [62].
In addition, Andreasen et al. evaluated the extension of the effect of its functionalization in terms of distance from the implant site. The authors evaluated the performance of titanium coated with HA/βTCP functionalized with HyA or reinforced with poly-D,L-lactic acid (PDLLA) in femoral defects in comparison with allograft from healthy sheep. The study takes into consideration, for the quantitative measure, two defined regions of interest (ROIs): ROI-1 close to the implant and ROI-2 close to the host bone, both having an approximate width of 500 µm. The two types of functionalizations gave rise to a bone formation and mechanical fixation similar to the allograft group given that no statistically significant difference was observed in BIC parameter comparing allograft with the HA/βTCP, HA/βTCP-HyA and the HA/βTCP-PDLLA groups suggesting the capability of both strategies to induce bone formation comparably to the allograft [63].
Izquierdo-Barba et al. tested a silicon substituted hydroxyapatite (SiHA) functionalized with VEGF to coat a macroporous titanium implant, observing an higher extent of bone formation in TiSiHA-VEGF group in comparison with the other group (TiSiHA, Ti-VEGF or Ti scaffolds) suggesting that the combination of osteoconductive properties of hydroxyapatite with silica and its biochemical cue and angiogenetic properties sustained by VEGF were able to stimulate bone regeneration [64].
Other two innovative approaches have been found: the route followed by Alt et al., which used a nanoHA also functionalized with collagen type I (col-1) and the strategy adopted by Chandran et al., who combined in the same HA structure strontium and adipose derived mesenchymal stem cells. In the first research, the regeneration capability of nanocrystalline hydroxyapatite with (HA/col-1) or without collagen-type I (HA) was evaluated in comparison to empty defect. Also, alt:V identified defined ROIs: ROI1 corresponding to the initial defect region and ROI2 corresponding to a region with a distance of 1 mm to the initially created defect where host bone-defect/biomaterial interface was present. Comparable new bone formation in ROI1 was measured for both HA and HA/col-1 group in comparison to the empty defect group, while in the ROI2, the highest BV/TV and the smallest Tb. Sp. were obtained for HA in comparison to the other groups. HA/col-1 yielded the highest connectivity density and the highest trabecular number. Surprisingly, the functionalization with collagen type-I (to enhance cells adhesiveness by transmembrane integrin receptors) did not induce a significant enhancement of performance in comparison with nanoHA, maybe suggesting a key role exerted by nanostructure in this experimental set-up [65,66]. On the contrary, Chandran S., obtained the highest BV/TV values in defects treated with the synergistic functionalization of HA with the therapeutic and regulating effect Strontium and the osteogenic efficacy of adipose derived mesenchymal cells (ADMSCs) in comparison with HA, Sr-HA and ADMSCs-HA [66].

Clinical Studies
The clinical studies included in the review were five: 2/5 used ceramics for hip arthroplasty, while 3/5 studies used ceramic sticks or cement for spinal surgery. There was no mention on the micro or nanoscale of ceramics, as well as no testing of the functionalization of the ceramics (Table A3 in Appendix A).
Papers by Aro and Lee reported results of using ceramic coated prosthesis for hip primary replacements, both using stem prosthesis with an HA coated neck [67] or entirely HA coated [68].
In both studies, groups of patients implanted with non-HA-coated stems have not been reported; so far conclusions about the efficacy of using ceramic coatings to improve prosthesis osteointegration in OP patients cannot be drawn.
Aro et al. performed analyses during a 2-year single-centre prospective non controlled study on 39 female patients affected by osteoarthritis (OA) and requiring hip arthroplasty. Also, bone status was assessed by dual-energy X-ray absorptiometry (DEXA). RSA analysis (rotation, translation of the stem and osteointegration and clinical assessments (Harris hip and WOMAC scores) assessed that low BMD and ageing adversely affected initial stability of HA-coated implants and delayed osseointegration [67]. Lee et al. retrospectively evaluated 87 cementless long-stem prostheses extensively coated with HA in osteoporotic patients (mean T score −3.0, range, −2.5 to −6.3) affected by intertrochanteric fractures. Radiological and clinical analysis detected cortical porosis in the 37.5% of cases, acetabular erosion in 4.2%, heterotopic ossification in 6.3% while no subsidence, no failure of leg length equalization and no osteolysis, suggesting that HA coated long stems relieve pain, restore function, provide long-term stability with few complications comparable or better than using cemented prosthesis [68].
The other three clinical studies considered ceramic based cements or sticks for augmentation in spinal surgical procedures, such as kyphoplasty [69] or interbody fusion [70,71]. The studies of Shin et al. and Jang et al. have a comparator control arms consisting of empty controls rather than a clinically accepted control material.
In a prospective comparative study, Shin and colleagues applied HA sticks for pedicle screw augmentation in 22 OP (T value ≤ −2.5) and non-osteoporotic (T value > −2.5) patients affected by degenerative lumbar spine diseases and undergoing to interbody spinal fusion surgery. Results at 2 years highlighted that insertion with HA sticks significantly increased Torque values than without HA insertion in both OP and non-osteoporotic patients with significant higher values in the non-osteoporotic group than in the OP one. There were differences in bone sclerosis between HA and control groups, regardless of the presence of OP, while no differences in loosening events and clinical assessments have been observed. The authors concluded that HA stick augmentation enhanced initial pedicle screw fixation strength in OP patients.
Jang et al. retrospectively evaluated 34 osteoporotic patients (T-score of −2.5 or less) affected by spondylolisthesis and requiring transforaminal lumbar interbody fusion with polyethyetherketone (PEEK) cages: some patients received an additional screw augmentation with HA cement. Analyses conducted postoperatively up to the 2-year follow-up showed that in the group of patients augmented with HA cement there were no significant changes in radiologic parameters, differently from the non-augmented ones. Although not significantly, the visual analogue scale (VAS) and fusion rate were superior in HA cement treated patients. The authors concluded that screw augmentation with HA cements may be a useful tool in OP patients.
In a small cohort non-controlled retrospective study, Klein et al. [69] performed 24 kyphoplasties in 11 OP patients using CaP cement, formed by micro-crystalline calcium-deficient HA. They followed patients up to 3 years, assessing that the intraosseous cement volumes slightly decreased every year; in only one case cement was found in the spinal canal without occurrence of any side effect. Vertebral stability increased significantly in the postop; pain and mobility scores significantly improved 2 years and 1 year after implantation, respectively. The authors concluded that CaP cement could be a safe and effective material with clinical and structural results comparable to poly methyl methacrylate (PMMA)-based materials also for the treatment of osteoporotic vertebral fractures.

Discussion
The burden of OP fractures is increasing, especially in population over 60 years old, imposing a significant economic impact on society. As recently reported by the International Osteoporosis Foundation, the estimated number of hip fractures worldwide will reach the 6.26 million in 2050 with a projected increase by 240% in women and 310% in men [72]. The cost associated with the fracture treatment in the US is around $17.9 billion per annum while in the EU equates to €24 billion, even if considering the indirect costs, as long-term care and fracture prevention therapies, the amount rises to €37 billion per year [73].
Osteoporotic bone fracture represents a challenge intervention as the reduced mineral density, the bone micro-architectural deterioration and mechanical and structural modification of bone properties faced the surgeon and patient with a complex situation which compromises the achievement of a stable implant fixation and osseointegration [74]. Currently, in the clinical scenario, cements like PMMA or CaP represent the most commonly used biomaterials to treat osteoporotic fractures, especially at the spine level, in addition to metallic prostheses and osteosynthesis devices, nevertheless, new strategies are emerging to achieve better results in terms of osteointegration in compromised conditions.
In the present systematic review, the role of functionalization of ceramic materials or coatings with organic or inorganic molecules on osteointegration process in osteoporotic/osteopenic bone was evaluated from the preclinical and clinical point of view. The key role of the osteoconductive effects of ceramic materials on implant osteointegration in OP has already been reported, showing a better outcome in terms of BIC percentage and mechanical push-out test of CaP coated materials in comparison to non-coated implants [16].
The interface between bone and implant is a complex microenvironment, in which different "state" of the bone coexist: native bone, with an equilibrated homeostasis, peri-implant bone, which is driven to the onset of regeneration process and the newly formed bone. The deeper knowledge of bone structure and of the mechanisms of bone regeneration had helped to understand how to improve material design, at the micro and nanometric level, to obtain better and faster osteointegrative performance, also in terms of biomechanical competence [75].
Materials topography and geometries surely influence bone response and should balance the different forces (compressive, tensile and shear forces) which play a role in implant integration.
Increasing material roughness is a consolidated strategy to enhance osteointegration, as irregularity on the surface improve bone to implant contact and seems to guarantee a better biomechanical anchorage [76].
In the case of ceramic materials, it is well established that biological properties (osteoinductive and osteoconductive abilities) are also related to the synthesis process, in particular density, grain size, micro and macroporosity, pore dimension and interconnection, surface and bulk chemistry. For instance, some authors found that a decrease in ceramic particle size is related with a decrease in the inflammatory reaction [77,78]. Laquerriere et al. demonstrated the correlation between the secretion of inflammatory cytokines and ceramic particles sintered at high temperature [79]. The osteointegrative process is also critically dependent on surface charge and wettability, as well as nanotopography and microporosity.
Starting from this background and considering the increasing interest in the field of biomaterials functionalization, this review provides a picture of the currently studied and used molecules exploited to functionalize ceramic materials or coatings.
Most of the functionalization found in the present review involved changes in the chemical structure of ceramic materials by means the ionic substitution/enrichment with relevant ionic species (Sr 2+ , Mg 2+ , Zn 2+ , SiO 3 2+ , SiO 4 2− ). Through a process of a co-precipitation, the composition, energy and/or surface charge of the material are changed with a substantial modification of the physicochemical properties and the bioactivity of the ceramic material. This approach leads to a larger, effective functionalized area and 3D distribution of trace elements throughout the bulk material. Even small amounts of these ionic species substitutes in the structure of ceramics exert a significant effect on thermal stability, solubility, osteoclastic and osteoblastic responses, degradation and bone regeneration [53,54,[80][81][82][83][84].
Another interesting and unusual element adopted as inorganic components in the functionalization was lithium: after an initial observation of beneficial effect on bone status in patients undergoing lithium therapy for mental disorders, lithium proved to promote osteogenic differentiation, via activation of the Wnt signalling pathway, inhibiting glycogen synthase kinase-3beta (GSK-3β) [85].
Always through a process of co-precipitation or chemisorption, CaP biomaterials can be functionalized with bisphosphonates (i.e. ALN, RA or ZOL), the major class of antiresorptive drugs used in clinics to inhibit bone loss thus preventing fracture occurrence. In fact, the local use of BPs is adopted to increase bone density preventing bone loss thank to its anti-resorptive capacity and seems to have also a positive effect in modulating the timing of ceramic dissolution, which influences not only bone growth but also the release of loaded drugs or molecules [51,52,62,[86][87][88][89].
Chemical or physical changes at the titanium implant surfaces realized with alkali treatment, acid etching or anodic oxidation serves for acceleration and promoting the CaP coating adhesion. Physical immobilization (loading, dipping or adsorption) or chemical covalent immobilization represents the main functionalization strategies adopted to provide biological cues as drugs, growth factors, peptide or cells at the CaP coating surfaces. [4] Alongside these types of "traditional" functionalization, focused on implementing the early adhesion of the host bone to the implant and therefore aimed at favouring the primary stability, more innovative approaches have been investigated. Examples are the functionalization with components of the ECM of bone like collagen type I or fibronectin involved in the modulation of cells behaviour through sequences recognized by cells integrin receptors or hyaluronic acid, glycosaminoglycan with a role in tissue support, lubrication and viscoelasticity modulation.
Bone tissue is also responsive to specific growth factors or hormones. Thus, the localized and controlled delivery of these bioactive molecules enhance the efficiency of bone regeneration as demonstrated by the functionalization with BB-1, a mutant BMP with enhanced osteogenic capacity comparable to that of BMP-2 or with VEGF or rhNELL-1 an osteoinductive factor with osteogenic and anti-osteoclastic properties.
Among the exploited growth factors, very interesting was the functionalization with BDNF. The choice of BDNF lays in the increasing evidence of its involvement in bone fracture healing, assessed in rodent models. Besides the role played during inflammatory response and enhancing angiogenic process, BDNF acts via TrkB-cRaf-ERK1/2-Elk1 signalling pathway enhancing the activity of osteoblasts and osteoblast-like cells, influencing ALP, BMP and OPN levels [90]. Others used anabolic factors comprise the application of statins that came from the evidence that patients undergoing therapy for hypercholesterolemia seemed to have a better bone status, probably thanks to the action on an enzyme in the mevalonate pathway downstream of HMG-CoA reductase, involved in bone loss. Despite indications in this sense are not so clear, it is instead assessed the ability of statins to increase VEGF and BMP-2 levels, being involved in bone regeneration processes [91]. Melatonin functionalization is also tested as the relationship between melatonin and bone health has been the object of discussion for many years. Many in vitro studies evidenced trophic effects on bone metabolism and cells, promoting osteoblast differentiation and increasing levels of bone formation markers. In addition, the inhibitor effect of Mel on inducible nitric oxide synthases (iNOS), which is involved in the onset of osteoporotic processes, make this hormone particularly suitable to be used in condition of compromised bone metabolism [92]. Finally, icariin is a well-established drug of traditional Chinese medicine, used for different disorders, including those affecting joints and for the treatment of osteoporosis. Icariin acts by increasing OPG/RANK ratio, TGF-β1, insulin growth factor 1 (IGF-1) and bone related proteins as osteopontin, osteocalcin, bone sialoprotein, enhancing bone formation [93].
All these functionalization strategies arisen from the present systematic review follow the compelling need to limit bone resorption and at the same time to stimulate bone deposition around ceramics. In fact, the foreign body response to differently reabsorbable materials, such as ceramics, is a great concern particularly in a compromised microenvironment, as in OP. When a prosthetic material is implanted, the process of osseointegration occurs through the establishment of the foreign body equilibrium [94] that has to be maintained as long as possible. Within bone, macrophages exert a pivotal role on one hand in driving the transition between the M1 inflammatory phenotype in the early reparative phase and the M2 anti-inflammatory phenotype in the later stages [94] and secondly being precursors of osteoclasts.
Ceramic coatings under loading conditions are subjected to wear and fragmentation, leading to the generation of particles. If the foreign body equilibrium is lost, repeated events of phagocytosis of ceramic particles maintain and strengthen the M1 inflammatory phenotype stimulating an inflammatory cascade, involving different cells (mainly monocytes/macrophages, polymorphonuclear cells and multinucleated giant cells) and growth factor and cytokine production (IL1, IL8, TNF-a, MMPs) that ultimately cause osteoclast activation, bone resorption and peri-implant osteolysis [95].
Inflammatory, cellular and biological responses to ceramics are particularly relevant in an OP microenvironment, in which the balance between osteoblasts and osteoclasts is already altered and the foreign body equilibrium is more difficult to maintain. Thus, the need of ceramics that are able to modulate and limit bone resorption is compelling.
Two papers take into consideration a functionalization strategy based on the use of autologous cells, OPG gene-modified BMSCs and ADSCs, respectively. Considering the ever-increasing use of cellular adjuvants in many fields of regenerative medicine, it would be expected more advanced studies on this innovative biotechnology. In both studies, the functionalization with mesenchymal stem cells play a key role in bone regeneration and repair processes thanks to their osteogenic differentiation and paracrine and trophic activities [37,49]. However, it must be taken into consideration that this type of functionalization would be related to an allogenic harvesting of BMSC or ADSC in clinical practice to obviate at the osteoporotic condition. Therefore, concerns exist related to harvesting procedure and morbidity for the patient, as well as concerns pertaining to the translation of this type of strategy to clinics relying on the need of an authorized cell factory to manipulate cells with inherent direct and indirect costs.
Surprisingly, the use of nanostructured ceramic materials is exploited in few papers: that is, Kettenberger et al. evaluated a nHA combined with hyaluronic acid hydrogel with or without ZOL [24], while Alt. et al. studies nHA with or without collagen [65]. In both studies the authors did not observe statistically significant differences between functionalized groups and nHA, thus suggesting a significant role played by the nanostructured size of ceramic in the osteoporotic condition capable of obtaining comparable results to the functionalized groups ( Figure 2). inflammatory cascade, involving different cells (mainly monocytes/macrophages, polymorphonuclear cells and multinucleated giant cells) and growth factor and cytokine production (IL1, IL8, TNF-a, MMPs) that ultimately cause osteoclast activation, bone resorption and peri-implant osteolysis [95].
Inflammatory, cellular and biological responses to ceramics are particularly relevant in an OP microenvironment, in which the balance between osteoblasts and osteoclasts is already altered and the foreign body equilibrium is more difficult to maintain. Thus, the need of ceramics that are able to modulate and limit bone resorption is compelling.
Two papers take into consideration a functionalization strategy based on the use of autologous cells, OPG gene-modified BMSCs and ADSCs, respectively. Considering the ever-increasing use of cellular adjuvants in many fields of regenerative medicine, it would be expected more advanced studies on this innovative biotechnology. In both studies, the functionalization with mesenchymal stem cells play a key role in bone regeneration and repair processes thanks to their osteogenic differentiation and paracrine and trophic activities [37,49]. However, it must be taken into consideration that this type of functionalization would be related to an allogenic harvesting of BMSC or ADSC in clinical practice to obviate at the osteoporotic condition. Therefore, concerns exist related to harvesting procedure and morbidity for the patient, as well as concerns pertaining to the translation of this type of strategy to clinics relying on the need of an authorized cell factory to manipulate cells with inherent direct and indirect costs.
Surprisingly, the use of nanostructured ceramic materials is exploited in few papers: that is, Kettenberger et al. evaluated a nHA combined with hyaluronic acid hydrogel with or without ZOL [24], while Alt. et al. studies nHA with or without collagen [65]. In both studies the authors did not observe statistically significant differences between functionalized groups and nHA, thus suggesting a significant role played by the nanostructured size of ceramic in the osteoporotic condition capable of obtaining comparable results to the functionalized groups ( Figure 2). A non-negligible result that emerges from the revision work is related to the significant discrepancies in the experimental settings adopted, even in the context of papers adopting the same animal model: namely, OP induction, implantation surgery, confirmation of osteoporosis development, anatomical site of implant, number of animals and type of materials. Preclinical studies were representative mainly of post-menopausal and glucocorticoid induced secondary osteoporosis and only one study evaluated male OVX. Thus, there is the lack of testing models that mimic OVX and osteopenia secondary to ageing or harmful lifestyles, such as sedentary habits or alcohol and tobacco consumption. According to the author, these aspects, in a rapidly ageing A non-negligible result that emerges from the revision work is related to the significant discrepancies in the experimental settings adopted, even in the context of papers adopting the same animal model: namely, OP induction, implantation surgery, confirmation of osteoporosis development, anatomical site of implant, number of animals and type of materials. Preclinical studies were representative mainly of post-menopausal and glucocorticoid induced secondary osteoporosis and only one study evaluated male OVX. Thus, there is the lack of testing models that mimic OVX and osteopenia secondary to ageing or harmful lifestyles, such as sedentary habits or alcohol and tobacco consumption. According to the author, these aspects, in a rapidly ageing population, should be carefully evaluated. A wide range in the waiting time between the OVX and implantation surgeries has been found: from 3 to 12 weeks for rats and from 3 to 10 months for sheep. These relevant differences make the comparison between studies difficult. In addition, in some papers, evaluations for the confirmation of the onset of osteoporotic state development were not performed. Despite ovariectomy surgery now being a standardized and recognized procedure, the measurement of BMD is mandatory to properly evaluate obtained results (Figure 3). population, should be carefully evaluated. A wide range in the waiting time between the OVX and implantation surgeries has been found: from 3 to 12 weeks for rats and from 3 to 10 months for sheep. These relevant differences make the comparison between studies difficult. In addition, in some papers, evaluations for the confirmation of the onset of osteoporotic state development were not performed. Despite ovariectomy surgery now being a standardized and recognized procedure, the measurement of BMD is mandatory to properly evaluate obtained results (Figure 3). Rodents still represent the most used type of animal model; a quick overview on the preferred site of implants shows, as expected, that the majority of tested materials formulations are placed in long bones (usually femurs) and vertebrae (mainly in ovine model), classical anatomical sites evaluated in cases of OP. Moreover, maxillofacial implants are less frequent, with 2 studies in which the mandible was the implant site, although recent literature highlights that oestrogen loss strongly influences bone resorption in the mandible [96] and clinical evidence have shown that the depth of mandibular incisure can be evaluated for early detection of OP [97]. Critical size defects are also performed in calvaria bone, despite even in this case where there are only few works about but this site is more and more used for studies related to OP [98,99] (Figure 4).
In almost all of the evaluated studies microtomography, histology and histomorphometry concur in the evaluation of osseointegration: bone volume/total volume (BV/TV), trabecular bone parameters, bone to implant contact and bone density represent the main evaluated parameters. Also, mechanical tests are represented, underlying an interest not only in bone regeneration but also in the evaluation of the quality of newly formed bone and implant stability.
To summarize, in all the considered approaches, the improvement of osseointegration parameters is probably due to the synergistic effect provided by the dissolution of the ceramic material and the anti-resorptive or anabolic effect of elements, drugs and growth factors. It remains to verify the extent, both spatially and temporally, of the effect induced by the functionalization strategies: only 3 papers take into consideration the extent of the observed effect, reporting a loss of significance of a few millimetres away from the implant [62,63,65].
Alongside the innovative functionalization strategies and approaches proposed in the preclinical field, the clinical scenario did not present any of the aforementioned approaches. Most of the clinical studies were retrospective (3/5) in small groups of patients with a follow-up that ranged between 1 and 3 years. The investigated ceramic implants were commercially available products without functionalization and without any information about the micro-or nano-topography, probably due to the registered trademarks. All the studies agree that in osteoporotic patients, CaP materials or coating restore function, enhancing initial implant fixation strength and that low BMD, as well as ageing, affects the initial implant stability and delayed osseointegration, even for ceramic coated stems. Rodents still represent the most used type of animal model; a quick overview on the preferred site of implants shows, as expected, that the majority of tested materials formulations are placed in long bones (usually femurs) and vertebrae (mainly in ovine model), classical anatomical sites evaluated in cases of OP. Moreover, maxillofacial implants are less frequent, with 2 studies in which the mandible was the implant site, although recent literature highlights that oestrogen loss strongly influences bone resorption in the mandible [96] and clinical evidence have shown that the depth of mandibular incisure can be evaluated for early detection of OP [97]. Critical size defects are also performed in calvaria bone, despite even in this case where there are only few works about but this site is more and more used for studies related to OP [98,99] (Figure 4).
In almost all of the evaluated studies microtomography, histology and histomorphometry concur in the evaluation of osseointegration: bone volume/total volume (BV/TV), trabecular bone parameters, bone to implant contact and bone density represent the main evaluated parameters. Also, mechanical tests are represented, underlying an interest not only in bone regeneration but also in the evaluation of the quality of newly formed bone and implant stability.
To summarize, in all the considered approaches, the improvement of osseointegration parameters is probably due to the synergistic effect provided by the dissolution of the ceramic material and the anti-resorptive or anabolic effect of elements, drugs and growth factors. It remains to verify the extent, both spatially and temporally, of the effect induced by the functionalization strategies: only 3 papers take into consideration the extent of the observed effect, reporting a loss of significance of a few millimetres away from the implant [62,63,65].
Alongside the innovative functionalization strategies and approaches proposed in the preclinical field, the clinical scenario did not present any of the aforementioned approaches. Most of the clinical studies were retrospective (3/5) in small groups of patients with a follow-up that ranged between 1 and 3 years. The investigated ceramic implants were commercially available products without functionalization and without any information about the micro-or nano-topography, probably due to the registered trademarks. All the studies agree that in osteoporotic patients, CaP materials or coating restore function, enhancing initial implant fixation strength and that low BMD, as well as ageing, affects the initial implant stability and delayed osseointegration, even for ceramic coated stems.

Conclusions
The illustrated results clearly show that functionalization strategies significantly improve osseointegration, representing a viable option in the treatment of osteoporotic fracture. Nevertheless, this improvement is probably affected by a limited range in terms of extension and further in-depth investigations are surely mandatory to clarify these aspects, beyond all the biological mechanisms driving tissue responses to functionalized implants. Although the clinical application of these advances is yet to be fully finalized, functionalization certainly promises to represent a winning strategy to improve the primary stability of implants and to ensure a quicker functional recovery of patient life quality.
Funding: This research was partly funded by National Funding Organizations (Ministero della Salute-IMH) under the frame of EuroNanoMed III Project "Next generation antibacterial nanostructured osseointegrated customized vertebral replacement-NANOVERTEBRA" Joint Transnational call for proposals (JTC 2018) and partly by Ricerca Corrente to the IRCCS-Istituto Ortopedico Rizzoli.

Conclusions
The illustrated results clearly show that functionalization strategies significantly improve osseointegration, representing a viable option in the treatment of osteoporotic fracture. Nevertheless, this improvement is probably affected by a limited range in terms of extension and further in-depth investigations are surely mandatory to clarify these aspects, beyond all the biological mechanisms driving tissue responses to functionalized implants. Although the clinical application of these advances is yet to be fully finalized, functionalization certainly promises to represent a winning strategy to improve the primary stability of implants and to ensure a quicker functional recovery of patient life quality.
Funding: This research was partly funded by National Funding Organizations (Ministero della Salute-IMH) under the frame of EuroNanoMed III Project "Next generation antibacterial nanostructured osseointegrated customized vertebral replacement-NANOVERTEBRA" Joint Transnational call for proposals (JTC 2018) and partly by Ricerca Corrente to the IRCCS-Istituto Ortopedico Rizzoli.

Conflicts of Interest:
The authors declare no conflict of interest.        [41]