The impact of Zn-doped synthetic polymer materials on bone regeneration: a systematic review

To repair bone defects, a variety of bone substitution materials have been used, such as ceramics, metals, natural and synthetic polymers, and combinations thereof. In recent decades, a wide range of synthetic polymers have been used for bone regeneration. These polymers have the advantages of biocompatibility, biodegradability, good mechanical properties, low toxicity, and ease of processing. However, when used alone, they are unable to achieve ideal bone formation. Incorporating zinc (Zn) into synthetic polymers has been considered, as previous studies have shown that Zn2+ promotes stem cell osteogenesis and mineral deposition. The purpose of this systematic review was to provide an overview of the application and effectiveness of Zn in synthetic polymers for bone regeneration, whether used alone or in combination with other biomaterials. This study was performed according to the PRISMA guidelines. A search of the PubMed, Embase, and the Cochrane Library databases for articles published up to June 2020 revealed 153 relevant studies. After screening the titles, abstracts, and full texts, 13 articles were included in the review; 9 of these were in vitro, 3 were in vivo, and 1 included both in vitro and in vivo experiments. At low concentrations, Zn2+ promoted cell proliferation and osteogenic differentiation, while high-dose Zn2+ resulted in cytotoxicity and inhibition of osteogenic differentiation. Additionally, one study showed that Zn2+ reduced apatite formation in simulated body fluid. In all of the in vivo experiments, Zn-containing materials enhanced bone formation. At appropriate concentrations, Zn-doped synthetic polymer materials are better able to promote bone regeneration than materials without Zn.


Background
Bone defects caused by various conditions, such as congenital malformation, trauma, tumor resection, and infection, not only cause great pain but also place tremendous pressure on healthcare systems [1]. Bone tissue engineering (BTE) emerging in recent decades now provides an option for the therapy of bone defects. The method involves combining biomaterials with donor cells to promote bone regeneration, and a variety of biomaterials have been used in BTE to repair and replace traumatized/damaged bone tissue, such as ceramics, metals, natural and synthetic polymers, and combinations thereof [2,3].
As a commonly used material both in conventional processing methods, like freeze-drying and solvent-casting, and emerging three-dimensional (3D) printing technology, a wide range of synthetic polymers have been applied for bone regeneration, including polylactic acid (PLA), polycaprolactone (PCL), poly (glycolic acid) (PGA), and their copolymers, such as poly (lactic-co-glycolic) acid (PLGA). Approved by the Food and Drug Administration, these polymers are considered biocompatible with natural tissues [4] and have several other advantages. For example, as synthetic materials, their composition, architecture, and physical properties are adjustable; also, these materials are highly reproducible. Moreover, these polymers are degradable, so they can provide mechanical support initially, before gradually degrading to make room for newly formed tissue [5,6]. However, although synthetic polymers can be osteoconductive, they usually do not achieve the desired effect of complete bone regeneration when used alone [4].
Zinc (Zn), an essential trace element, is usually found in skeletal tissue, and approximately 30% of Zn in the body is contained in bone tissue. Zn plays a significant role in the formation, development, mineralization, and maintenance of healthy bones [7]. Also, as a mediator of bone development and growth, Zn deficiency in humans can lead to diseases such as dwarfism, osteoporosis, and stunted bone development. Previous in vitro studies have demonstrated that Zn 2+ improves stem cell osteogenesis and enhances mineral deposition [8].
Thus, Zn incorporation is expected to improve the osteogenic ability of synthetic polymer materials, as the release of Zn 2+ may enhance the osteogenic differentiation of cells for accelerated bone regeneration [9].
However, there is no consensus on the precise role of Zn in these effects, and different types of cells seem to respond differently depending on the Zn 2+ concentration. In a study by Bertels et al., 0.04-0.08 mM of zinc sulfate (ZnSO 4 ) enhanced mineral nodule formation in swine adipose-derived stem cells (swine ASC) in osteogenic media; moreover, the response was biphasic: concentrations of ZnSO 4 above 0.08 mM were detrimental to cell growth [10]. And Tiffany et al. found that Zn 2+ in amounts below 0.04 mM still enhanced the cell number and metabolic activity of porcine adipose-derived stem cells (pASCs) [11]. In another study, Xiong et al. showed that 10.91-27.15 μM of Zn 2+ in cell culture medium significantly enhanced the proliferation and alkaline phosphatase (ALP) activity of mouse bone marrow-derived mesenchymal stem cells (mBMSCs). Similarly, high concentrations (128.58 μM) of Zn have been shown to significantly inhibit ALP activity [12].
In this systematic review, we describe the incorporation of Zn into synthetic polymer materials (whether used alone or in combination with other biomaterials), evaluate the effectiveness of these Zn-doped materials for promoting osteogenesis, and discuss the viable concentration of Zn 2+ for osteogenesis in different types of cells.

Objectives
The objective of this research was to review the literature on Zn-doped synthetic polymer materials, to provide an overview of the application and effectiveness of incorporating Zn into synthetic polymers to improve their osteogenic ability.

Guidelines
The PRISMA guidelines were followed in this research [13].

Inclusion and exclusion criteria
The following are the inclusion criteria: In vitro studies In vivo studies with animal bone defect models Zn-doped synthetic polymer materials used alone or in combination with other biomaterials Materials used in the form of membranes, scaffolds, disks, etc.

Published in English
The following are the exclusion criteria: Synthetic polymer materials not used No control group

Search strategy
The PubMed, Embase, and Cochrane Library databases were searched for studies about the application and effectiveness of Zn-doped synthetic polymer materials for promoting osteogenic ability, published at any time up to June 2020. The following keywords were used: "Zinc," "Polymers," and "Osteogenesis." Details of the search strategies are provided in Supplementary Materials Tables S1, S2, and S3.

Study selection and data collection
Studies were selected by two reviewers (Li R, Zhao X), who independently screened the titles, abstracts, and full texts of all retrieved articles. Disagreements were resolved by discussion or through consultation with a third reviewer (Wang S). Potentially suitable articles were then assessed according to the inclusion and exclusion criteria, and data were then extracted. A flow chart of the study selection process is shown in Fig. 1.

Quality assessment
Two other researchers (Zhu Y, Gu R) used the Methodological Index for Non-Randomized Studies (MI-NORS) scale to evaluate the risk of bias [14,15]. The revised and validated version of MINORS contains 12 items. Each item is scored from 0 to 2 (0, not reported; 1, inadequately reported; 2, sufficiently reported). The ideal total score is 16 for non-comparative studies and 24 for comparative studies. Disagreements regarding scores were resolved by discussion.

Study selection and characteristics
Of the 153 articles screened, 13 were deemed eligible for the review. In total, 28 articles were excluded due to being duplicates, and a further 55, 38, and 19 studies were excluded after screening the title, abstract, and full text, respectively. The contents of the 13 included articles are systematically summarized in Tables 1 and 2, and the results of the quality assessments are listed in Table 3. All included studies had scores above 16 on the MINORS scale, indicating a low risk of bias. In these studies, synthetic polymers were typically used in conjunction with other biomaterials. Nine of the studies were conducted in vitro, three were in vivo, and one included both in vitro and in vivo experiments. Three of the studies were performed on New Zealand rabbits (tibia or skull) [26][27][28], and one was performed on Sprague-Dawley (SD) rats (femur) [24]. The in vitro studies were concerned with cell proliferation and viability, and osteogenic differentiation, assessed using MTS, MTT, and CCK-8 assays, and ALP activity, RT-PCR, immunofluorescence staining, Alizarin Red S (ARS) staining, and calcium content assay, respectively.

Results of individual studies
To assess the effect of Zn-doped synthetic polymer materials on bone regeneration, proliferation/viability and osteogenic differentiation data were obtained, and in vivo osteogenesis evaluations were performed, as discussed above.   [22].

Cell osteogenic differentiation assessments
Of the 13 articles, 10 assessed osteogenic differentiation based on ALP activity, RT-PCR, immunofluorescence staining, ARS staining, and calcium assay. One study used all of the abovementioned evaluation methods [17], and the others used at least one of the techniques. Overall, Zn-containing materials significantly promoted osteogenic differentiation compared to the control groups without Zn. Amiri et al. fabricated a Zn 2 SiO 4 -PES-PEG scaffold and showed the osteogenic differentiation rate of human mesenchymal stem cells (hMSCs) on this scaffold was increased compared to that of PES-PEG fibrous scaffolds [17]. Li et al. demonstrated that, consistent with cell proliferation results, the PAA-0.025M and PAA-0.05M groups exhibited higher ALP activity and more calcium nodules compared to the PAA and PAA-0.1M groups [22]. Interestingly, Rajzer et al. found that PCL-A2Zn5 (PCL combined with 49CaO-5ZnO-6P2O5-40SiO2 bioactive glass [BG]) membranes promoted ALP expression, whereas in simulated body fluid (SBF), apatite formation on the surface of the PCL-A2 (PCL combined with 40SiO2-54CaO-6P2O5) membranes was markedly increased compared to pure PCL and PCL-A2Zn5 membranes [20].
It is worth mentioning that some of the studies added other metal ions to the polymer materials besides Zn 2+ and thus achieved better results with respect to promoting bone regeneration. Deng et al. reported that a silver (Ag)/Zn-codecorated SPEEK surface showed enhanced cell proliferation and osteogenic differentiation  their results showed that the expression of bone morphogenetic protein 2 (BMP-2) was higher in the PLGA/ CPC-Si/Zn scaffolds [24].

In vivo experiments
In the in vivo experiments, bone defect sites were created in the tibia, femur, and skull, typically in New Zealand rabbits or SD rats (Fig. 2). All of the studies showed that Zn-containing materials can play a role in promoting bone formation, consistent with in vitro studies. It should be noted that Liang et al. demonstrated that PLGA/CPC-Si/Zn scaffolds yielded a substantial increase in the amount of regenerated bone volume over the PLGA/CPC-Zn scaffolds [24].

Discussion
The unique advantages of synthetic polymers include biocompatibility, biodegradability, good mechanical properties, and low toxicity; additionally, their shape, porosity, and mechanical properties can be well defined and controlled, and they can be stably mass-produced [29]. However, hydrophilia deficiencies and a lack of bioactivity compromise the ability of synthetic polymers to NPs nanoparticles, Dox doxycycline, PLGA poly (lactic-co-glycolic acid), CPC calcium phosphate cement facilitate biomaterial-host interactions. Furthermore, they typically show poor antibacterial properties, and there is some concern that their degradation products generate an acidic environment conducive to inflammation [5]. Therefore, synthetic polymers are commonly combined with other biomaterials to obtain twodimensional or 3D scaffolds that promote the desired cell behavior and tissue regeneration. Some of the 13 studies included in our review support the conclusion that the addition of BGs can address the abovementioned issues. In the study of Rajzer et al., an SBF bioactivity test demonstrated that the presence of BG on PCL membranes induced mineralization, thus indicating its bioactivity [20]. As a solution to the problem of acidic products, Oh et al. showed that the ions dissolved from BGs neutralize the acidic environment caused by polymers, and reported significantly higher expression of ALP and osteocalcin in samples containing BG and, especially, ZnBG [16]. To improve antibacterial performance, Bejarano et al. incorporated BG doped Each item is scored 0 (not reported), 1 (inadequately reported), or 2 (sufficiently reported). The global ideal score is 16 for non-comparative studies and 24 for comparative studies Fig. 2 In vivo experiments with animal bone defect models. a Skull defects in rabbits [27,28]. b Tibia defects in rabbits [26]. c Femur defects in SD rats [24] with copper (Cu) and Zn into poly (D, L-lactide) (PDLL A). The results showed that the Cu-and Zn-doped BG improved antibacterial activity [18]. When used alone, synthetic polymers usually cannot achieve the desired effect of complete bone regeneration. In recent years, biodegradable metals (BMs) have gradually become a hotspot in the field of biomedical materials used in BTE because of their good biocompatibility, degradability, and appropriate mechanical properties. Among BMs, Zn has received extensive attention due to its satisfying biological properties in promoting bone regeneration [30].
Thus, incorporating Zn into the synthetic polymers combines the advantages of both and continues to receive much attention. Previous studies have indicated that Zn can regulate cells, which stimulates osteoblastogenesis and attenuates osteoclastogenesis. In the process of bone formation, Zn regulates the secretion and expression of osteogenic markers, such as ALP, and the deposition of minerals [31,32]. However, the role of Zn in the process of bone regeneration depends on its content. In the selected articles, Zn is usually incorporated into the polymers as a composite or coating (Fig. 3). The content of Zn in the composite materials ranged from 0.11 to 2.9 wt%; the results indicated that, in this range, Zn exerts a satisfactory bone-promoting function. While in the Zn-containing coatings, the concentration of Zn did not show consistency, generally within the range of 20 wt%, as the surface area, thickness, and density of the coatings may also count greatly in the Zn-release process. Nevertheless, it can be concluded that Zncontaining coatings have viable osteogenic effects. Moreover, several studies have indicated that Zn-coating bone implants enhance osseointegration [33,34].
Previous studies have discussed the possible toxic effect of Zn on cells. Some researchers pointed out that in the case of BGs,~5 mol% Zn doping is the upper limit before cytotoxicity occurs. Additionally, only the highest concentration (5 mg/L) elution extracts inhibited the growth of mouse embryonic fibroblasts (MEFs) [35][36][37]. However, Neščáková et al. [7] showed that Zn-doped BGs (~8 mol% of ZnO) did not exhibit cytotoxicity towards MG-63 or MEF cells; this can be explained by the relatively low Zn 2+ concentration released into the medium (1.2 mg/L). Thus, it can be inferred that, in terms of the likely effects of Zn on the biological behavior of cells, the release concentration of Zn 2+ in aqueous solutions is a more accurate indicator than the proportion of Zn in materials.
In aqueous solutions, different cells respond differently to the Zn 2+ concentration (Table 4).
Rajzer et al. found out that in SBF, BG containing Zn showed less apatite formation on the surface compared to BG without Zn [20]. This could be explained that in SBF, Zn may influence the kinetics of hydroxyapatite (HA) formation and retard HA nucleation [45]. By binding to the active growth sites of HA, Zn 2+ prevents its nucleation [46] (Fig. 4). Moreover, as the Zn 2+ content increases, the deposition rate of HA decreases. Previous studies have pointed out that, at low concentrations, the released Zn 2+ may stimulate osteoblast differentiation, osteogenic differentiation of mesenchymal stem cells (MSCs), and extracellular matrix (ECM) mineralization in vitro [47][48][49], whereas at high concentrations, Zn 2+ reduces ECM mineralization and may cause cytotoxicity [30,31]. It can be concluded that, with appropriate concentrations and release behavior, the addition of Zn to a bone scaffold is likely to promote bone regeneration.
The mechanism by which Zn promotes bone formation has been partially explored but remains unclear. Zhu et al. used pure Zn disks and human mesenchymal stem cells (hMSCs) to show that intracellular Zn 2+ triggers Ca 2+ responses by activating the cyclic adenosine monophosphate protein-kinase A (cAMP-PKA) pathway, followed by the mitogen-activated protein kinase (MAPK) pathway. The Gαq-PLC-AKT pathway is also activated by Zn 2+ ; the activity in all of these pathways enhances cell growth and differentiation, ECM mineralization, and differential regulation of genes [50]. Similarly, Park et al. suggested that ZnSO 4 exerts osteogenic effects on human bone marrow-derived mesenchymal stem cells (hBMSCs) by activating RUNX2 via the cAMP-PKA-CREB pathway [51]. Fathi and Farahzadi demonstrated that, in electromagnetic fields, ZnSO 4 activated the extracellular signal-regulated protein kinase (ERK) 1/2, PKA, and Wnt/β-catenin signaling pathways to promote the osteogenesis of adipose tissue-derived MSCs [52]. Yamaguchi et al. showed that ZnSO 4 attenuated the nuclear factor-kappa B (NF-κB) pathway activation induced by tumor necrosis factor-alpha (TNF-α), to promote osteoblast mineralization and suppress osteoclast differentiation, and could also mitigate the transforming growth factor-beta/bone morphogenic protein-2 (TGF-β/BMP-2)-induced inhibitory effect of TNF-α on the activation of Smad [39,40]. In addition, there have been several studies of Zn-containing composite materials. Vimalraj et al. revealed that Zn-silibinin complexes regulated the miR-590/Smad7 signaling pathway to enhance osteoblast differentiation [53]. Fernandes et al. Osteoclast 10 −12 -10 −4 mol/L for cell activity Higher than 10 −4 to promote the proliferation of TRAP-positive cells [41,42] Osteoblasts and primary murine bone marrow stromal cells Lower than 10 −9 M for proliferation Lower than 10 −9 M for osteogenic and adipogenic differentiation [42,43] rBMSCs 10 −5 mol/L to achieve the best osteogenic effect - [43,44] Vascular Endothelial cells 1.1 mg/L for proliferation 2.7 mg/L to cause cytotoxicity [34,35] Vascular smooth muscle cells 60-80 μM for cell adhesion, spreading, proliferation, and migration 80-120 μM for the same cellular functions [37,38] Abdomen RAW264.7 10-250 μM to suppress osteoclastogenesis - [39,40] incorporated Zn and citrate into HA nanoparticles and demonstrated osteogenic differentiation of BMSCs in the absence of osteoinductive factors, with the functioning regulated, at least in part, by activation of the canonical Wnt pathway [54]. Using Zn-modified calcium silicate coatings, Yu et al. showed that the TGF-β/Smad signaling pathway plays a significant role in regulating the osteoblastic differentiation of rat bone marrow-derived pericytes [55]. Despite these findings, the mechanism by which Zn-doped synthetic polymer materials promote osteogenesis has not been fully elucidated. Some of the 13 studies in this review used multiple ions. Deng et al. demonstrated that Ag nanoparticledecorated and Ag/ZnO-codecorated SPEEK effectively inhibit the reproduction of Gram-positive and Gramnegative bacteria. Moreover, Ag/ZnO-codecorated SPEEK substrates were better able to enhance the biocompatibility and osteodifferentiation of MG-63 cells, likely due to the combined effect of micro-/nanoscale topological cues and Zn induction [19]. In the study of Telgerd et al., Cu not only acted as an angiogenic agent, but also enhanced angiogenesis in vitro and increased the proliferation rate of endothelial cells [21]. Liang et al. showed that silicon (Si) promotes vascularization; they integrated Si-Zn and PLGA microspheres into CPC scaffolds, in which Si 4+ was released at a faster rate than Zn 2+ to initially promote angiogenesis and, later, osteogenesis. This was attributed to the biodegradable PLGA microspheres, which allowed for the successional release of Si and Zn. As the sequential vascularization and osteogenesis corresponded to the natural process of bone defect restoration, the PLGA/CPC-Si/Zn group showed better bone regeneration than the PLGA/CPC-Zn group [24]. Therefore, the synergistic effect of different metal ions on the biological properties of synthetic polymers is a topic worthy of further investigation and could be explored using Zn-doped synthetic polymer materials.
Based on the MINORS, the studies in this review were of satisfactory quality. However, no clinical studies were obtained through our database searches, indicating that synthetic polymers have not yet been applied clinically in humans. Meanwhile, the lack of standardization among the studies prevented us from conducting a meta-analysis.
We believe that the main significance of this systematic review is to prove the addition of Zn to synthetic polymers promotes bone regeneration; moreover, the viable ranges of Zn 2+ concentrations for different cell types were demonstrated. However, additional research is necessary to uncover the mechanism by which Zndoped synthetic polymer materials promote bone Fig. 4 Zn 2+ prevents HA nucleation by binding to its active growth sites regeneration and other potential clinical applications. Finally, the effect of Zn addition on other properties (e.g., mechanical, degradation, and biocompatibility properties) should be examined further.

Conclusion
Synthetic polymers are commonly used in bone regeneration; however, their ability to promote bone formation is limited when used alone. The introduction of Zn may address this issue. In this systematic review, an overview of the application and effectiveness of Zn incorporation into synthetic polymers is presented. The results showed that, with appropriate concentrations and release behavior, Zn-containing synthetic polymers have the potential to promote bone regeneration. However, the mechanism of action and feasibility for clinical application require further study.