Large bone defects caused by trauma or therapeutic resection are difficult to treat. Often, the defect void is filled with bone graft to provide structural integrity, osteo-conduction, osteo-induction, or a combination thereof. Autograft bone harvested from the patient yields the best clinical outcomes. However, the amount of autograft that can be harvested is limited, and donor site morbidity is a significant clinical concern. Alternative approaches may include using the Masquelet method to generate additional tissue for grafting or employing limb-lengthening procedures to avoid grafting altogether [1–4]. Allograft and synthetic bone graft materials are commonly used to treat large bone defects [5]. Allograft is generally abundant and available in different physical forms that can be tailored to the required need. However, the ability of allograft or synthetic bone graft to promote osteogenesis within a bone defect is significantly less than that of autograft [6, 7].

Zinc is an essential nutrient that concentrates in bone and appears to have one or more physiological roles in the skeleton [8]. Previous studies found that dietary Zn deficiency leads to reduced levels of Zn in bone and can impair adolescent growth in humans and animal models [9–13]. Clinical studies have found that elevated serum Zn levels correlate with increased bone mineral density and reduced fracture risk [14]. Conversely, patients with osteoporosis were found to have reduced serum Zn levels and reduced bone tissue levels of Zn [15, 16]. Animal models and in vitro studies have also found that Zn can inhibit osteoclast activity and stimulate osteoblast activity [17–20].

Zinc can also promote bone regeneration when applied to sites of bone healing. Using a rat femur fracture model, local application of Zn to the fracture site significantly improved healing as shown by enhanced callus cellularity, increased callus levels of insulin-like growth factor-1 and vascular endothelial growth factor, increased percentages of callus cartilage and bone, and increased callus mechanical properties, demonstrating the potential therapeutic use of Zn for bone regeneration [21–23]. Local application of Zn with autograft improved posterolateral spinal fusion in a rat model [24]. Several studies have also noted that incorporating Zn into synthetic materials can enhance the osteogenic activity of those materials [25–27]. A recent experiment also found that adsorbing Zn to morselized, processed human allograft significantly improved osteogenesis in critically-sized rat femoral defects as compared to non-adsorbed control allograft [28].

Zinc also has anti-microbial properties. Using zinc acetate, Atmaca et al. [29] found that soluble Zn inhibited Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) growth at concentrations above 2.2 mM but had no effect on the growth of Pseudomonas aeruginosa (P. aeruginosa). Gugala et al. [30] also found that approximately 2 mM Zn could inhibit the growth or biofilm formation of S. aureus, but was less effective against P. aeruginosa or Escherichia coli (E. coli). Inclusion of Zn into bioactive glass at 1.2% Zn by weight or hydroxyapatite at 1.6% Zn by weight inhibited the growth of Streptococcus mutans (S. mutans) and S. aureus, respectively [31, 32]. Thus, treating bone allograft with Zn may also provide a beneficial anti-microbial effect during and after allograft preparation.

The improved osteogenic capacity of the Zn-treated allograft suggests that binding of Zn to the allograft can be adjusted to improve clinical performance. To that end, this study measured the effects of Zn concentration, incubation time, potentially competitive divalent cations, and different Zn salts on Zn-binding to human allograft. By examining adsorption parameters that affect Zn binding to allograft, we aim to better understand how allograft Zn treatment can be optimized for future in vivo testing. The potential use of Zn-binding to allograft to reduce bacterial burden was also investigated.

Processed human cortical cancellous bone allograft was obtained from a commercial vendor (SpineFrontier, Beverly, MA, USA). Tissue banks must obtain consent from the donor or the donor’s family before collecting any tissue. The allograft was pulverized and sieved to obtain 0.5 to 1 mm size granules, which were used in all subsequent experiments. Micro-spin filters [0.2 µm pore size polyvinylidene fluoride (PVDF) membrane, 850 µL capacity, polypropylene housing] in 2 mL microcentrifuge tubes were purchased from Analytical Sales and Services, Inc. (Flanders, NJ, USA). PVDF midi-spin filters (0.2 µm pore size membrane, 4 mL capacity, polypropylene housing) in 7 mL tubes were purchased from Ciro Manufacturing Corp. (catalog number BPV02, Deerfield Beach, FL, USA). Anhydrous zinc chloride and zinc acetate dihydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Zinc picolinate was purchased from Spectrum Chemical Mfg. Corp. (New Brunswick, NJ, USA). The different Zn salts were dissolved in Tris-buffered saline (TBS) that consisted of 50 mM Tris-Cl and 150 mM NaCl, pH 7.4.

Staphylococcus aureus, subspecies aureus, strain NCTC 8532 (S. aureus) was purchased from the American Type Culture Collection (Manassas, VA, USA). S. aureus was cultured at 37°C in Difco Nutrient Broth media or agar plates (Fisher Scientific, Pittsburgh, PA, USA). Colonies of S. aureus from agar plates were used to inoculate 3 mL of Difco Nutrient Broth, that were cultured overnight at 37°C with shaking. The overnight cultures were first diluted to an optical density (OD₆₀₀) of 0.1 [equal to 10⁷ colony forming unit (CFU) per mL] in fresh Difco Nutrient Broth and then to 10³ CFU/mL in TBS in preparation for treating bone allograft. Only fresh, overnight cultures were used for experiments.

The experimental results indicate that the amount of Zn binding to the allograft is not dependent on ZnCl₂ concentration (from 0.1% to 1% w/v) when treatment time is relatively short (< 15 min, Figure 1). However, Zn binding to allograft is dependent on ZnCl₂ concentration when treatment time is longer (1 hour or more) and the ZnCl₂ solution is > 0.1% ZnCl₂ (Figure 1) [28]. This suggests that Zn binds to allograft bone through more than one modality. As bone is composed of both organic and mineral components, the fast, concentration-independent binding and slow, concentration- and time-dependent binding of Zn may reflect differential Zn binding affinities between the organic and mineral components of bone or between surface binding of Zn to bone hydroxyapatite versus Zn substitution of calcium within the hydroxyapatite crystal [34]. In support of the former, a study from Fuierer et al. found that Zn bound to the surface of synthetic, non-sintered hydroxyapatite, most likely as a zinc phosphate surface precipitate, while Haumont detected Zn in the calcifying cartilage of rat growth plates and the osteoid (non-mineralized bone protein matrix) of canine Haversian canals [35, 36]. We found that even after 1 hour of incubation in 0.5% ZnCl₂, binding of Zn to the bone allograft occurred primarily on the surface (Figure 5). The presence of Tissue Non-specific Alkaline Phosphatase (TNSALP) at sites of calcifying cartilage and mineralizing osteoid would contribute to Zn localization at these sites since Zn is a co-factor for TNSALP [37, 38]. However, phosphate produced from TNSALP enzymatic activity at sites of calcifying cartilage and osteoid may amplify local Zn levels by precipitating Zn as zinc phosphate. Interestingly, Fuierer et al. [36] also found that Zn binding to the surface of hydroxyapatite inhibited further hydroxyapatite crystal growth. Thus, controlling soluble levels of Zn at sites of active bone formation may be a means to regulate bone mineral deposition.

Differences in Zn binding to allograft from the different zinc salts were somewhat unexpected (Table 1 and Figure 2). The expectation was that the aqueous solubility of each zinc salt would correlate with the concentration of Zn²⁺ ions available to bind to the bone allograft. As expected, the low aqueous solubility of zinc picolinate leads to reduced binding of Zn to the allograft bone as compared to the highly aqueous soluble zinc chloride. In contrast, the moderately soluble zinc acetate produced more Zn binding to the bone allograft than the highly soluble zinc chloride. The difference between the rapid binding of Zn to bone allograft from zinc acetate versus zinc chloride is not known. However, the ability of Zn²⁺ to interact with water appears to be affected by how well the anion can buffer in order to prevent Zn precipitation [39]. Since the buffering capacity of acetate is much greater than chloride, perhaps this could account for the faster binding of Zn to bone allograft from a zinc acetate solution than a zinc chloride solution.

The mechanism by which Zn binds to the allograft is not known. Spadaro et al. [40] found that Zn bound to both the organic (collagen) and mineral phases of bone, indicating multiple mechanisms for sequestering Zn in bone tissue. Several trace metals are found in normal bone tissue, and among those trace metals, Mg and Zn are two of the more abundant elements [41, 42]. Mg and Zn dissolve as divalent cations. Thus, if Zn binding to the bone allograft was strictly through an ionic interaction, excess levels of Mg²⁺ should compete for Zn²⁺ binding. However, we detected no competitive effect of Mg on Zn binding even at an approximate 2.5-fold excess of Mg (0.5% w/v ZnCl₂ and 2.0% w/v MgCl₂-6H₂O, Figure 3). In addition, binding of Zn was independent of pH over the pH 6 to pH 8 range tested (Figure 4). This indicates that Zn binding to the allograft has more specificity than a simple ionic interaction.

In addition to improving the osteogenic activity of processed bone allograft [28], Zn binding to allograft also appears to reduce the ability of bacteria to attach to allograft or to remain viable when attached to the Zn-bound allograft. As shown in Figure 6, Zn-bound allograft was resistant to binding viable S. aureus, one of the most common pathogens associated with osteomyelitis [43]. While even 1 min of treating the allograft with 0.5% w/v ZnCl₂ reduced viable S. aureus binding, longer ZnCl₂ incubation times appeared to be more effective.

Use of Zn in orthopaedic applications is an area of increasing interest because of the positive effects of Zn on osteogenesis and because Zn can be used to make biodegradable metal fixation devices [8, 44]. Use of Zn as an allograft adjuvant to enhance bone regeneration offers significant advantages over other approaches. Unlike growth factors or other protein adjuvants, Zn should not be affected by standard sterilization procedures and would allow for convenient, room temperature storage of the Zn bound allograft. As noted, Zn is an essential trace mineral that is necessary for multiple molecular processes. An estimated 10% of the human proteome binds Zn [45]. Sufficient amounts of Zn are generally consumed through a normal diet, but oral supplements are commonly used. The recommended daily allowance for Zn consumption in the United States is 8 mg/day for women and 11 mg/day for men, with a recommended maximum of 40 mg/day. The LD₅₀ for oral Zn toxicity is 3 g/kg body weight [46, 47]. Zn is used therapeutically for topical and enteric applications. Zn is considered a generally recognized as safe and effective substance by the US Food and Drug Administration. In our prior work, we found that Zn bound to allograft at 0.4% w/w was effective in promoting bone formation, which would equate to 40 mg of Zn per 10 g of bone allograft [28]. At the 0.4% w/w dose level of Zn to allograft and given the apparent slow-release of Zn from allograft, use of Zn-bound allograft to treat skeletal injuries would be unlikely to produce any safety concerns.

The results of this study will be useful for determining how best to restore trace metals or other substances to bone allograft that are lost during processing. In addition, the results of this study indicate that trace substances do not necessarily bind to bone through simple ionic interactions with the hydroxyapatite mineral phase of bone. Whether the binding mechanism utilized to restore Zn levels to processed allograft is similar to how Zn normally binds to bone in vivo will require additional investigation. Still, the results of this study can be used to help optimize methods that will enhance the osteogenic activity and bacterial resistance of bone allograft to provide better clinical outcomes.