Antibacterial 3D bone scaffolds for tissue engineering application

Abstract

Open bone fractures are not only difficult to heal but also are at a high risk of infections. Annual cases of fractures which result from osteoporosis amount to approximately 9 million. Endogenously released nitric oxide (NO) has been shown to play a role in osteogenic differentiation in addition to eradicating infection against a wide variety of pathogens. In the current work, antimicrobial NO releasing 3D bone scaffolds were fabricated using S-nitroso-N-acetyl-penicillamine (SNAP) as the NO donor. During fabrication, nano-hydroxyapatite (nHA) was added to each of the scaffolds in the concentration range of 10–50 wt % in nHA-starch-alginate and nHA-starch-chitosan scaffolds. The mechanical strength of the scaffolds increased proportionally to the concentration of nHA and 50 wt % nHA-starch-alginate possessed the highest load bearing capacity of 203.95 ± 0.3 N. The NO flux of the 50 wt % nHA-starch-alginate scaffolds was found to be 0.50 ± 0.06 × 10⁻¹⁰ mol/min/mg initially which reduced to 0.23 ± 0.02 × 10⁻¹⁰ over a 24 h period under physiological conditions. As a result, a 99.76% ± 0.33% reduction in a gram-positive bacterium, Staphylococcus aureus and a 99.80% ± 0.62% reduction in the adhered viable colonies of gram-negative bacterium, Pseudomonas aeruginosa were observed, which is a significant stride in the field of antibacterial natural polymers. The surface morphology and pore size were observed to be appropriate for the potential bone cell growth. The material showed no toxic response toward mouse fibroblast cells.

INTRODUCTION

A bone injury or a bone defect is a result of lost bone integrity either due to an external physical force or due to a decrease in peak bone mass (osteoporosis). More than 75 million people are affected by osteoporosis in United States, Japan, and Europe every year.¹ Annual cases of fractures which result from osteoporosis amount to 8.9 million, that is, one osteoporotic fracture every 3 s. About 9 million osteoporotic fractures were reported globally in the year 2000, 51% of which were from the United States and Europe.² Musculoskeletal injuries, in fact, is the most common medical reason behind the failure of soldiers to be deployed in the battlefield.³ It is also the cause of 73% of total disability cases in the US army contributing to an annual compensation of $5.5 billion to affected soldiers.⁴ Some bone defects may arise from tumor trauma, or bone-related diseases and affect millions of lives worldwide.⁵ Open fractures are not only difficult to heal but are also at high risk of infections. Infection caused by microbial pathogens diverts the inflammatory response away from healing and hence delays the recovery. Deep infections of bone often occur in the joints and at the ends of long bones.⁶ Prevention of bone infections past surgeries require courses of systemic antibiotics and surgeries prior to bone grafting.⁷ Unfortunately, even in the light of optimal health management, the chances of infection in open fracture is approximately 30% (of all the cases).^8,9

A biodegradable three-dimensional (3D) scaffold made from natural polymers serves as a temporary skeleton to accommodate new tissue growth in bone tissue engineering.¹⁰ These are particularly amenable to implantation and can be easily manufactured into desired shapes.^11,12 In the current study, nitric oxide (NO) releasing antibacterial 3D bone scaffolds were fabricated using chitosan-starch or alginate-starch as the base polymer and the mechanical strength was provided using the hydroxyapatite. A comparison was made between nHA-alginate-starch versus nHA-chitosan-starch with varying nHA concentrations for its compressive strength to refine what composition is best in terms of maximum load-bearing capacity. After finding the best composition of NO donor, SNAP was incorporated to it. Endogenous NO synthesized by NO synthase (eNOS) promotes bone development and healing. In addition, research has shown that NO helps in osteogenic differentiation of bone marrow mesenchymal cells (BMSC) in 3D silk scaffolds.¹³ However, its antibacterial potential as an alternative to antibiotics application during bone defects has not been studied yet. The NO released within the sinus cavities and macrophages functions as a natural antimicrobial agent to combat pathogen invasion in humans.¹⁴ Nitric oxide is a free radical gas with a short half-life that plays an important role in various biological processes. Moreover, the use of NO is unlikely to stimulate the production of resistant strains due to non-specific action, short half-life, and rapid reduction of microbial load life.^15–17

Owing to its biological activity and inherent bactericidal effect, chitosan has garnered great interest in bone scaffold applications.¹⁸ Alginate is another natural polymer which is frequently used for bone grafting due to its biocompatible, biodegradable, and hydrophilic nature.^19–21 Furthermore, the integration of starch in natural polymers can adequate mechanical properties and controlled degradation of bone scaffolds.²² Such biopolymers, when reinforced with a bioactive bone component, for example, ceramic fillers such as hydroxyapatite (HA), pose great bone tissue regeneration capabilities. Many studies have reported that addition of a calcium phosphate such as HA can enhance the biological properties of bone scaffolds due to its compositional similarity to natural bone.²² The bone implants containing higher concentrations of HA have been shown to have a better affinity toward bone in vivo over those with the smaller amounts of it.²³ In the past few years, HA has been extensively used in the bone cement formulations for repairing of the femur and craniofacial defects. The addition of nHA has been shown to increase the scaffold surface area due to the decrease in the pore size.²² Such an increase in the scaffold’s surface area favored by nHA has been shown to positively affect bone cells growth.¹⁸

While nHA, starch, alginate, and chitosan are well-known materials used for bone tissue engineering, utilization of exogenous NO donor in bone scaffold fabrication is a new and unexplored area of research. The primary objective of this work is to explore the possibility of integrating a nitric oxide donor, SNAP in 3D bone scaffolds to prevent bacterial infection without causing any cytotoxicity to the mammalian cells. In the current study, various concentrations of nHA (10–50 wt %) were added to starch and blended with either alginate or chitosan to formulate a solution for 3D bone scaffolds. The study first tested nHA-starch combination with alginate or chitosan for its compressive strength filter what composition is best in terms of maximum load bearing capacity. Thereafter, the 3D scaffold formulation showing maximum compressive strength were incorporated with the SNAP and characterized further for the NO release kinetics and effect of SNAP incorporation on the morphology of the scaffolds. Finally, the ability of the bone scaffolds to prevent infection was tested against common infectious agents such as gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa. The 3D scaffold is hypothesized to provide a matrix to restore damaged bone function and prevent infection without causing any cytotoxicity to healthy mammalian cells.

MATERIALS AND METHODS

Materials

Alginic acid sodium salt, chitosan, and reagent grade hydroxyapatite (calcium phosphate tribasic, MW: 502.31 g/mol) were obtained from Sigma-Aldrich, Co. (St. Louis, MO). Cornstarch was purchased from ACH Food Companies, Inc. (Memphis, TN). Sodium hydroxide and hydrochloric acid were obtained from Fisher Scientific (Fair Lawn, NJ). Glutaraldehyde and calcium chloride (anhydrous, granular ≤7.0 mm, ≥93%) were purchased respectively from Fischer Scientific (Fair Lawn, NJ) and Sigma-Aldrich, Co. (St. Louis, MO). The bacterial strains Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 5538) used in this were originally obtained from American Type Culture Collection (ATCC).

Methods

Synthesis of nano-hydroxyapatite.

The nHA particles can penetrate through the surface of bone and can aid in the remineralization of a decayed bone. Therefore, a standard protocol developed by Sundaram et al., was used to synthesize nHA particles from hydroxyapatite (HA).²² Hydroxyapatite solution (3.4 wt %) was dissolved completely in 150 mL of 0.1 M HCl using a magnetic stirrer. The 1 M NaOH was added dropwise to the beaker containing 3.4% (w/v) HA solution. The mixture was allowed to agitate continuously using a magnetic stirrer. As the reaction progressed, nano-crystals of HA started to precipitate in the beaker. The pH was readjusted to 6.8–7.2 using concentrated HCl. The resulting solution was stirred for 20 h to allow aging of nHA crystals. Then, the crystals were collected by centrifuging the solution for 20 min at 3000 rcf (Eppendorf Centrifuge 5702). The supernatant was discarded and the nHA pellet was stored at 4–8°C until used in the preparation of scaffolds.

Fabrication of nHA-starch-alginate and nHA-starch-chitosan composites.

Five different types of nHA-starch-alginate composites and nHA-starch-chitosan composites were prepared using different wt % of nHA: 10, 20, 30, 40, and 50 wt %. Firstly, 2% (w/v) solution of alginic acid (n = 5) was prepared by adding 1 g of alginic acid in 50 mL of deionized water (diH₂O) and stirred at 40–45°C for 3 h. Separately, 2% (w/v) solution of cornstarch (n = 5) was heated up to at 60–65°C while constantly stirring it for 4 h using a magnetic stirrer. As the starch solution became dense and thick, different wt % of nHA (10–50 wt %) were added to individual starch solutions and stirred for another 10 min at room temperature. The starch-nHA blend was added to alginate solution to get a formulation of nHA-starch-alginate with varying concentration of nHA in the range of 10–50 wt %. Similarly, the nHA-starch-chitosan blend was obtained using the same reaction condition except for dissolving 2% (w/v) solution of chitosan in 1 M acetic acid solution instead of diH₂O. The resulting formulations with varying levels of nHA-starch-alginate (10–50 wt % of nHA) and nHA-chitosan (10–50 wt % of nHA) were cast in molds and were frozen at −15°C to −35°C in the freezer for 24 h to get solid scaffold composites.

Cross-linking of the biopolymer composites.

A collection of 10, 20, 30, 40, and 50 wt % of nHA-starch-alginate and nHA-starch-chitosan formulations were cross-linked. The nHA-starch-alginate bone scaffolds were cross-linked with 2% CaCl₂ solution. 2% CaCl₂ solution was prepared by dissolving 4 g of CaCl₂ in 200 mL of distilled water. The nHA-starch-alginate bone scaffolds were cross-linked in 5% glutaraldehyde. The 5% glutaraldehyde was prepared by dissolving 10 mL of glutaraldehyde in distilled water volume made up to 200 mL. Once the crosslinking solution became homogenized, scaffolds were immersed in the solution to crosslink for 2–3 h. Figure 1 represents the stepwise fabrication process of 3D bone scaffolds. Based on the mechanical strength results the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) was added in the formulation of the scaffolds showing maximum compression strength (Sections “Mechanical testing” and “SNAP synthesis and incorporation in bone scaffolds”).

FIGURE 1.

Fabrication process of 3D bone scaffolds: nHA-starch-alginate and nHA-starch-chitosan scaffolds. At step D, the NO donor, SNAP, was added to the 3D scaffolds to provide antimicrobial characteristics while the control 3D scaffolds were fabricated without SNAP. Subsequently the emulsion was frozen below −15°C for at least 24 h. Afterward, scaffolds were crosslinked and frozen again below −15°C overnight. Finally, 3D scaffolds were freeze-dried in a lyophilizer.

Creation of porous morphology using freeze-drying methods.

In order to serve as a vehicle to deliver the drug, promote cell interactions and retain cells at a specific site and prevent infections for faster bone healing, a porous biodegradable composite is needed as a scaffold.¹⁰ In addition, the drug release profile and the cell growth are strongly influenced by the pore size. Hence, it is crucial to execute appropriate processing and drying method to get the desired pore size. A greater surface area provides a platform for cell adhesion and in return allows cell proliferation, which sequentially assists bone tissue formation. In addition, it facilitates exchanges of nutrients and gasses for faster cells growth and hence enhanced healing.

An innovative yet simple technique of lyophilization was used to freeze dry the cross-linked 3D scaffolds. To create microscopic pores in the scaffolds, freeze-drying was carried out using a LABCONCO FreeZone 4.5 Plus freeze dryer. After crosslinking the 3D scaffolds with their respective chemical cross-linkers the scaffolds were frozen again in the freezer (−15°C to −30°C) overnight. Afterward, they were placed in a sealed beaker attached to a freeze drier and dried for 22 ± 2 h at 0.5 mBar vacuum and −84°C. The images of completely processed freeze-dried bone scaffolds are shown in Figure 2.

FIGURE 2.

Representative images of the final 3D bone scaffolds after complete cross-linking and lyophilization: A. nHA-starch-chitosan and B. nHA-starch-alginate. Right before, lyophilization the chitosan scaffold and alginate scaffolds were crosslinked with 5% glutaraldehyde and 2% CaCl2 solution in water.

Mechanical testing.

Completely dried scaffolds (n = 3 samples with l = 3 cm, w = 2 cm, h = 2 cm) were used to measure mechanical strength in terms of compressive force using an Instron model 5545. The plateau in the load versus strain curve was observed at compression ratios between 0.15 and 0.2 signifying a transition from the elastic to plastic deformation regime. No preload was used. A force perpendicular to the direction of the scaffold was applied and the cross-head speed was set to 1 mm/s. Force and distance were recorded and analyzed to obtain compressive strength.

A comparison was made between nHA-alginate-starch and nHA-chitosan-starch with varying nHA concentrations for its compressive strength to filter what composition is best in terms of maximum load-bearing capacity. The bone scaffolds composition with best mechanical strength was then screened to finally add 10 wt % SNAP during the fabrication process.

SNAP synthesis and incorporation in bone scaffolds.

SNAP synthesis.

S-nitroso-N-acetylpenicillamine (SNAP) was synthesized using recommendations from a previously reported method.^24,25 An equimolar amount of N-acetylpenicillamine (NAP) and sodium nitrite was added to a solution with 1:1 ratio of water and methanol containing 2 M H₂SO₄ and 2 M HCl. The resulting solution was stirred for 30 min. The precipitation of SNAP crystals was achieved by continuously aerating the reaction mixture for 4 h while the reaction vessel was cooled in an ice bath. The SNAP crystals were collected by filtering the solvent using a 70 mm Whatman^® filter paper and allowed to air dry overnight while the SNAP crystals were protected from light. The crystals’ purity was found to be greater than 95% using the Sievers Chemiluminescence Nitric Oxide Analyzer.

SNAP incorporation in the bone scaffolds.

The SNAP was incorporated in the bone scaffolds with the maximum compressive strength to provide it with NO releasing property. Briefly, 10 wt % SNAP was added to nHA-starch blend (prepared as shown in Section “Fabrication of nHA-starch-alginate and nHA-starch-chitosan composites”) and dissolved using a magnetic stirrer at room temperature. The resulting solution was added to completely dissolved 2% (w/v) alginate solution. The nHA-starch-alginate formulation containing NO donor, SNAP, was cast in a mold and placed in the freezer at −15°C to −35°C for 12–16 h overnight. The frozen material was cross-linked using 2% CaCl₂ for 2–3 h and the cross-linked scaffolds were frozen again overnight. Finally, the scaffolds were freeze-dried; using LABCONCO FreeZone4.5 Plus freeze dryer at ≤0.5 mBar vacuum and −84°C.

Morphological analysis.

The surface morphology and micro-structure of the scaffolds were examined using scanning electron microscopy (SEM) (FEI Inspect F FEG-SEM) in triplicates for each of the 3D bone scaffolds of nHA-starch-alginate and of nHA-starch-chitosan containing 10–50 wt % nHA. Freeze dried scaffolds were mounted on a metal stub with double-sided carbon tape and sputter coated with 10 nm gold–palladium using a Leica EM ACE200 sputter coater. Images were taken at an accelerating voltage of 20 kV at 100× magnification.

In addition, cross-sectional images of the 50 wt % nHA-starch-alginate scaffold with SNAP were taken using EVOS XL microscope at a resolution of 10×.

NO flux measurement of SNAP incorporated nHA-starch-alginate scaffolds.

Nitric oxide released by the 50 wt % nHA-starch-alginate composites were measured using a Sievers Chemiluminescence Nitric Oxide Analyzer (NOA) model 280i (Boulder, CO). The NOA can selectively measure NO through the reaction of NO with oxygen plasma, giving it the ability to reduce interference from molecules such as nitrates and nitrites. Portions of the dried SNAP-nHA-starch-alginate scaffolds were placed in the NOA cell with 3 mL of PBS with EDTA in triplicates (n = 3). Nitrogen was aerated into the PBS to facilitate the release of NO from solution at a rate of approximately 200 mL/min as recommended by NOA manufacturer. Following the initial measurement, the scaffolds were maintained at 37°C in a water-jacketed incubator (Thermo Fisher Scientific, Waltham, MA) in PBS. After 24 h, the scaffold was removed from the PBS solution and placed in the NOA cell with fresh PBS with EDTA. Release rates from the scaffolds were normalized on a per mass basis. The NO release rate per weight of the bone scaffolds was reported as NO flux (×10⁻¹⁰ mol/min/mg).

Bacterial adhesion test.

A standard bacterial adhesion test was used to assess antibacterial properties of NO releasing scaffolds.^26,27 A single isolated colony of P. aeruginosa and S. aureus strain was picked from an LB agar Petri dish, introduced to 10 mL of liquid LB medium and incubated for 14 h at 37°C. The optical density of the culture was measured at 600 nm (O. D₆₀₀) using UV–vis spectrophotometer (Thermo Scientific Genesys 10S UV–Vis). The traces of LB medium were removed by centrifuging the bacterial culture for 7.5 min at 2500 rpm, the supernatant was then discarded and sterile phosphate buffer saline (PBS, pH 7.4) of the equivalent amount was added and centrifuged again for 7.5 min at 2500 rpm twice. The OD₆₀₀ of the culture in PBS was measured again using PBS as the blank and diluting the bacterial culture in PBS to get the concentration in the range of 10⁶–10⁸ colony forming units per ml (CFU/mL). Segments of bone scaffolds, with and without SNAP, were weighed and exposed to a 50 mL tube containing 10 mL bacterial culture of S. aureus suspension and P. aeruginosa (n = 3). These scaffolds were incubated for 24 h at 2500 rpm in 37°C environment. After the 24 h bacteria study, each of these bone scaffolds was placed in a new 50 mL sterile tube containing 10 mL of fresh PBS, followed by 60 s of mixing in the vortex to remove loosely bound bacteria. Subsequently, the resulting bacterial suspension was serially diluted (10⁻¹ to 10⁻⁵) and plated in petri dishes with LB agar medium and incubated at 37°C for 24 h. After incubation, colony forming units per weight of the bone scaffolds (CFU/mg) were counted and compared with control and SNAP bone scaffolds to detect their efficacy to inhibit bacterial adhesion on the surface of bone scaffolds. Percentage bacterial inhibition was calculated using the formula below

$$\%\text{ Bacterial inhibition}=\frac{\text{CFU}/\text{mgin control samples}-\text{CFU}/\text{mgin test sample}}{\text{CFU}/\text{mgin control samples}}\times 100$$

Cytotoxicity test.

The NO releasing scaffolds were tested for the absence of cytotoxic response toward mammalian cells by modifying a recommended cell cytotoxicity assay.²⁷ The leachates from the samples (n = 7) were collected by putting 10 mg of sample per 10 mL of Dulbecco Modified Eagle’s Medium (DMEM) for 24 h at 37°C.

For cell culture, a cryopreserved vial containing 3T3 mouse fibroblast cells (ATCC-1658) was thawed and cells were cultured in a 75 cm² T-flask with complete DMEM medium. The cells were incubated in humidified conditions of 37°C with 5% CO₂. DMEM was replaced intermittently every second day and cells were observed daily for the absence of contamination. After reaching a confluency of 80% (adherent culture) cells were detached from the surface by enzymatic trypsinization (trypsin with 0.18% trypsin and 5 mM EDTA). Finally, the cells were counted using a hemocytometer with 0.4% trypan blue (dye exclusion method). Thereafter, in a 96 well plate (cell culture grade), 100 μL of 5000 cells/ml were seeded in each of the wells (7 wells for each sample type) and incubated for 24 h. As per the manufacturer’s recommendation, to each of the wells, 10 μL of the leachate from control (starch-alginate) and NO releasing biopolymer (SNAP-alginate) was added. Cells put in an incubator (5% CO₂, 37°C) were allowed to respond to the leachate for 24 h. Thereafter, 10 μL of the WST-8 solution (Sigma-Aldrich) was added and cells were incubated further for 24 h. WST-8 is converted to a formazan (an orange-colored product measured at an absorbance of 450 nm) in the presence of dehydrogenase enzyme secreted by live cells only. The relative viability (%) of the mouse fibroblast cells as a response to the leachate was measured using the formula below.

$$\%\text{ Cell viability}=\frac{\text{Absorbance of the test samples}}{\text{Absorbance of the control samples}}\times 100$$

Statistical analysis

Statistical data is expressed as a mean ± standard error of the mean. Comparison of means using student’s t-test: two samples assuming unequal variance were used to analyze if there was a statistical difference between the data for SNAP-incorporated versus control bone scaffolds. Values of p < 0.05 were considered statistically significant and were reported for all the experimental results unless otherwise mentioned.

RESULTS AND DISCUSSIONS

Biomaterials that can imitate the natural template for bone cells growth in terms of structure and composition can act as a potential candidate for bone tissue engineering applications. In the current study, we developed 3D bone scaffolds by combining nHA with natural polymers: alginate and chitosan and incorporated an NO donor in the scaffolds. In the past, the bactericidal effect of NO has been shown against a wide variety of pathogens: virus, bacteria, and fungus.^28–32 Besides its highly effective antibacterial nature, NO has crucial roles to play in bone growth, angiogenesis, tissue healing, and remodeling, which ensures faster recovery.³³ After determining the 3D scaffolds with maximum compressive strength between nHA-chitosan-starch and nHA-alginate-starch, SNAP was incorporated in the scaffolds with best load bearing capacity. Therefore, the NO release kinetics, SEM, antibacterial potential and cytotoxicity is tested and presented with only the scaffolds with maximum compressive strength.

Mechanical testing of the bone scaffolds

Compressive strength refers to the local stress maximum after the linear elastic region of the curve³⁴ which is one of the important functions of a bone scaffold to provide sufficient mechanical support temporarily to withstand in vivo loading and stresses.³⁵ The compressive strength of the scaffold should be retained until the tissue engineered transplant is fully remodeled by the host tissue and can assume its structural role. The maximum load bearing capacity of bone scaffolds should be high to provide proper load transfer at the site of implantation. A recent study showed that bone healing capacity increases in direct proportion to the amount of mechanical force applied.³⁶

During fabrication, nHA was added to each of starch-alginate and starch-chitosan solutions in the concentration range of 10–50 wt %. The cross-linking of the nHA-starch-alginate mix was achieved via two different mechanisms. First, during the covalent cross-linking of alginate with calcium ions present in the calcium chloride (cross-linker)²² and second, during the freeze-drying process. Removal of water promotes interchain cross-links in polysaccharides as in the case of proteins.³⁷ In general, there is more collapse of pore structures in freeze-dried samples because of processing at low temperature that results in less dense interchain cross-linking. However, due to the addition of nHA, the 3D scaffolds could not collapse and hence resulted in an improved compressive strength.

For the 3D scaffolds made with nHA-alginate-starch, the maximum load bearing capacity increased proportionally with the amount of nHA added to them as expected (Figure 3). In a previous study, a proportional increase in scaffolds strength with respect to nHA concentration has been shown.³⁸ The alginate-based scaffolds with 10 wt % nHA showed the least load bearing capacity at 74.0 ± 4.3 N as compared with 50 wt % nHA-starch-alginate scaffolds, which could tolerate a load of 203.9 ± 36.6 N before deformation. The 20, 30, and 40 wt % nHA-starch-alginate scaffolds corresponded to maximum load bearing capacities of 109.3 ± 10.0, 109.5 ± 14.3, and 122.2 ± 7.6 N, respectively. However, with the nHA-starch-chitosan, the compressive strength did not vary proportionally with nHA. Instead, with 20 wt % nHA, it exhibited the highest load bearing capacity which then slightly decreases and then remained constant for 30–50%. The nHA-starch-chitosan-based scaffolds (with 10–50 wt % nHA) showed maximum load-bearing capacity in the range of 111.5 ± 14.4 to 167.6 ± 52.2 N. Since both alginate and chitosan-based scaffolds were freeze-dried, the reason for the change in the mechanical strength might be due to covalent cross-linking which is material-dependent. In addition, many other factors such as the concentration of components, type of cross-linking agents, cross-linking time, composition of the solvent mixture, and cross-linking temperatures might affect the final mechanical strength. Overall, 50 wt % nHA-starch-alginate scaffolds showed the highest strength (203.9 ± 36.6 N) among all bone scaffolds and hence was chosen to incorporate 10 wt % SNAP and further characterization. In the past, it has been shown that the ultimate load to failure of and above 69.56 ± 4.74 N resulted in faster bone healing and thus 50 wt % nHA-starch-alginate scaffolds with highest compressive strength is expected to perform the best among all other scaffolds.³⁶ Furthermore, using the 50 wt % nHA can be advantageous with respect to other bone healing attributes, such as efficient bonding with host’s bone tissue and osteoconductive behavior, important for bone cells growth.³⁹

FIGURE 3.

Completely freeze-dried scaffolds were used to measure mechanical strength in terms of compressive force (N) using an Instron model 5545. The nHA was added at a concentration range of 10–50 wt % in both alginate (nHA-starch-alginate) and chitosan (nHA-starch-chitosan) scaffolds. Overall, 50 wt % nHA-starch-alginate scaffold showed highest strength among all bone scaffolds. Statistical data is expressed as mean ± standard error of the mean of n = 3 samples. Values of p < 0.10 were considered statistically significant.

Nitric oxide flux analysis of 3D bone scaffolds

Nitric oxide released from the 50 wt % nHA-starch-alginate scaffolds was measured using Sievers Chemiluminescence Nitric Oxide Analyzer (model 280i) under simulated physiological conditions (37°C, 3 mL PBS, pH 7.4). A representative release profile from the scaffolds is shown in Figure 4A. An initial burst of NO was seen prior to maintaining a steady release rate after approximately 30 min. Initial NO release rates from the scaffolds were 0.5 ± 0.06 × 10⁻¹⁰ mol/min/mg (Figure 4B). After 24 h in PBS at 37°C, the alginate scaffold continued to release NO at a rate of 0.23 ± 0.02 × 10⁻¹⁰ mol/min/mg. The theoretical lifetime of the NO release by the scaffold is extrapolated to be approximately 9 days when averaging the NO release rate over the first 24 h. The risk of infection is highest at the time of implantation and the pathogens can proliferate at the site of implantation. The bacteria are able to colonize a biofilm over the implant that is resistant to the therapeutic agents. Therefore, bone scaffolds that can control bacterial infection during the first week of implantation hold great therapeutic promise. The NO release over the first week may allow for adequate preliminary healing of the area and prevent immediate infection. The key point here is that preventing infection is most important in the first 1 week after implantation and thereafter, the 3D scaffolds can continue to support bone growth even in the absence of SNAP. However, if prolonged NO release is needed beyond 1-week, possible steps to improve the lifetime of the NO release may include increasing the overall NO donor concentration, as well as altering the composition of the scaffold material or crosslinking NO donor with the base polymer.

FIGURE 4.

Real-time NO release profile of SNAP incorporating 50 wt % nHA-starch-alginate scaffold. The comparative NO flux from SNAP incorporated 50 wt % nHA-starch-alginate bone scaffolds in the beginning day 0 (0 h) and day 1 (24 h). The theoretical lifetime of the scaffolds is extrapolated to be 8.6 days by averaging the release rate over the first 24 h. The flux corresponds to the lower end of physiological NO flux range, that is, 0.5–4.0, 10–10 to 0.23 ± 0.02 × 10–10 mol/min/mg. Statistical data is expressed as the mean ± standard error of the mean of n = 3 samples. Values of p < 0.05 were considered statistically significant.

In the past, controlled NO release from polymeric materials has been demonstrated by incorporating the NO donors into materials with low water uptake, whereas materials with high water uptake exhibit a large burst of NO release, where the NO supply is quickly depleted.^40,41 For instance, SNAP has been shown to release NO for greater than 18 days at physiological levels when incorporated into hydrophobic synthetic polymers at a concentration of 10 wt %.^24,40 These materials have been used in medical device coating applications, such as urinary and vascular catheters, and lend themselves to relatively low surface areas of release compared with those of porous scaffolds, where the surface area per unit weight (per mg) can be orders of magnitude higher. The more the surface area per mg is increased, the more the water uptake and hydrophilicity of the material will impact the NO release kinetics. While increasing the concentration of SNAP within the polymer matrix will aid in the stability and long-term release capabilities of the polymeric matrix through localized crystal formation of the NO donor, there are also negative impacts on the physical properties of the material.^24,42 In essence, the flexible fabrication model of the 3D bone scaffolds presented in the current study can be modified depending on the extent of the injuries by altering the type of polymers, NO donors, and their respective concentrations.

Surface morphology of 3D bone scaffolds

The morphology of a bone scaffold is another important property considered for replacement of bone to allow growth of bone cells. In addition, the strength of the 3D scaffolds can be adjusted to match site-specific requirements by manipulating overall porosity. In order to serve as a vehicle to deliver the drug, promote cell interactions and retain cells at a specific site and prevent infections for faster bone healing, a porous biodegradable composite is needed as a scaffold.¹⁰ In addition, the drug release profile and the cell growth are strongly influenced by the pore size. A greater surface area provides a platform for cell adhesion and in return allows cell proliferation, which sequentially assists bone tissue formation. In addition, it facilitates exchanges of nutrients and gasses for faster cells growth and hence enhanced healing.

Morphologies of the 50 wt % control nHA-alginate-scaffolds, as well as those containing SNAP, were examined under SEM. The addition of SNAP to the scaffold appeared to result in a decrease in the layering of the scaffold, possibly due to decreased cross-linking time. There were no observed regions of SNAP crystallization on the surface of the scaffolds. This combined with the steady NO release indicates that the SNAP is homogeneously mixed throughout the scaffold. The morphological analysis recorded by SEM is shown in Figure 5A,B. Previous reports have shown that the scaffolds with high porosity and interconnection provide the desired amount of space for cell seeding, growth and metabolites exchange.^43,44 The SEM analysis in the present study showed that the NO releasing bone scaffolds have a highly interconnected porous structure. The structural collapse in freeze-dried biomaterial is common; however, SEM analysis for the 50 wt % nHA-alginate-starch-based 3D scaffolds showed the uniform organization of pores within the scaffolds possibly due to prior screening that was done in selecting the optimized formulation in terms of maximum load for scaffold fabrication. Therefore, there was no pore structure collapse in 50 wt % nHA-starch-alginate scaffolds which synchronizes with the observations made in the mechanical strength testing. Another reason might be the addition of cornstarch. The cornstarch used in this study was thermally stabilized through starch gelatinization procedure, which gives strong interchain linkage when homogeneously mixed with alginate and chitosan gels. Additionally, nHA was added to improve the mechanical strength of the scaffolds. By increasing the mechanical strength, pore structure collapse during freeze-drying was eliminated as observed during SEM analysis. This is in line with the observation made from the cross-sectional images of freeze-dried scaffolds that shows a continuous and uniform matrix of the biopolymer materials (Figure 6A,B). In addition to these observations, no surface shrinkage was observed in the 3D scaffolds (Figure 2). The pore size based on SEM analysis were reported to be less than 1 mm. Different bone types have different requirement of pore sizes due to differences in their location, size, strength, and function. In general, tissue engineering scaffolds with a pore size in the range of 20–1500 μm are commonly used.^10,45,46 Pore size greater than 300 μm is recommended for promoting angiogenesis at the site of osteogenesis.⁴⁷ In addition, the highly interconnected pores in the NO releasing 3D bone scaffolds are expected to facilitate exchanges of nutrients and gasses for faster cells growth and hence enhanced healing which is in line with the earlier published work.^43,44 In the past also, studies have shown that starch-based bone scaffolds with nHA can support the growth of osteoblast cells in a 2 weeks study.⁴⁸

FIGURE 5.

Surface Electron Microscopy (SEM) images of A. Control 3D bone scaffold and B. 3D bone scaffold with SNAP. There were no observed regions of SNAP crystallization on the surface of the scaffolds. The highly interconnected pores are one of the desired characteristics of a bone scaffolds needed for cells seeding, gaseous exchange, nutritional transfer, and mass flow.

FIGURE 6.

Cross sectional view of freeze-dried 50 wt % nHA-starch-alginate scaffolds shows a uniform matrix of the biopolymer materials. The image on the left (A) is taken with 10-megapixel camera. The image on the right is taken using optical microscope (EVOS XL) at a resolution of 10×. The image B shows a pattern of uniform layer and void spaces in the scaffold. These images combined with Figures 2 and 5B give a holistic view of physical morphology of the bone scaffolds both internally and externally.

Bacterial inhibition on 3D bone scaffolds

Per American Academy of Orthopedic Surgeons (AAOS), patients with bone replacement often require another surgery to eradicate the bacterial infection, despite the use of antibiotics and preventative treatments. About 33% of the bone implantations results in infection even in the presence of optimal healthcare. Antibiotics are used at the site of implantation to control pathogenic population, but the never-ending issue of antibiotic resistance raises an alarming concern and questions its application as a long-term solution. If the infection prevails, it becomes necessary to remove the bone implant. Autografts are limited by the donor site morbidity while allografts can cause an undesired immune response in the host. These challenges have spurred the need for the development of polymeric 3D bone scaffolds with antibacterial properties. The 3D scaffolds are expected to only provide a matrix for bone cells to restore damaged bone function, but the problem of infection remains untreated and therefore slows down healing and prolong hospital stays increasing the overall cost of treatment. Gram-positive Staphylococcus aureus (S. aureus) remains the principal causal agent while the gram-negative Pseudomonas aeruginosa (P. aeruginosa) also contributes significantly toward bone infection that follows post-implantation.⁴⁹ Therefore, the study was carried out using common bacteria present in infected tissues S. aureus and P. aeruginosa.

The NO flux released by the 50 wt % alginate-starch-SNAP bone scaffolds ranged from 0.50 ± 0.06 × 10⁻¹⁰ to 0.23 ± 0.02 × 10⁻¹⁰ mol/min/mg over a 24 h period. This resulted in a 99.76% ± 0.33% reduction in a gram-positive bacterium, S. aureus and a 99.80% ± 0.62% reduction in the adhered viable colonies of gram-negative bacterium, P. aeruginosa as compared with alginate controls without SNAP (p < 0.05) in a 24 h study (Figure 7). This result is in line with the past reports including ours which have reported the antibacterial success of NO donors against pathogens such as S. aureus, S. epidermis, P. aeruginosa, S. epidermis, P. aeruginosa, E. coli, and A. baumanii.^{26,27,32,50–52}

FIGURE 7.

The antibacterial potential of NO releasing scaffolds after nHA addition to demonstrating that NHA addition does not affect the bactericidal potential of the alginate-SNAP biopolymeric matrix. The data is reported as a mean ± standard deviation for a sample size of n = 3 (for each sample type) with a p-value <0.05.

During bone surgeries, a joint spacer is often used to maintain space between the joint and the alignment. Space is filled with antibiotics that flow into the joint and surrounding tissue to eradicate the infection. The growing concern with the emerging antibiotics resistance bacterial strains and the associated mortality rises an alarming question about their use in future. According to a recent report in 2017 by Centers for Disease Control and Prevention, approximately 2 million people get infected with antibiotic-resistant bacteria, resulting in around 23,000 deaths per year in the United States alone. Nitric oxide-based approaches not only inhibit greater than 99% bacteria but can also provide an important alternative for the antibiotic based antibacterial treatment during bone surgeries. Nitric oxide acts via multiple mechanisms to kill the bacterial cells: membrane disruption, DNA breakage, and protein denaturation which ensures the high efficacy of killing.⁵⁴ In nature, the sustained release of endogenous NO endows macrophages with toxic activity against pathogens.⁵⁵ Moreover, the use of NO is unlikely to stimulate the production of resistant strains due to non-specific action, short half-life, and rapid reduction of microbial load life.^15–17 Another advantage of SNAP donor based strategies is its compatibility with other antimicrobial agents such as quaternary ammonium ions,²⁶ copper nanoparticles,²⁷ and silicone oil⁵⁶ and its easy blending in a variety of natural and synthetic polymers.^24,32

Absence of cytotoxic response toward mammalian cells

While inhibiting the bacterial infection at the site of medical device application is important, it is equally important that the material does not cause any toxic response toward the healthy mammalian cells. Very often, the leachates from the materials that can cause side effects to the nearby tissue and in some cases may have negative systemic implications too. Therefore, in the current study, leachates from the bone scaffolds were collected after soaking them in DMEM medium at 37°C for 24 h and then tested on mouse fibroblast cells for any potential cytotoxicity. Our results confirmed that while the material inhibited up to 99.8% bacteria, it did not show any toxic effect toward the mammalian cells (Figure 8). One plausible explanation for its non-cytotoxic nature can be as follows. SNAP breaks down to NAP and NO after cleavage of thiol bond. While NO is endogenous in nature it is expected to be harmless to mammalian cells in the physiological range, NAP is an FDA approved molecule used in the treatment of heavy-metal poisoning. Similarly, alginate and starch are derived from natural resources and are approved as edible food materials by FDA. Thus, any small amount of leaching from the composites was not expected to be toxic toward mammalian cells. In the past also, non-cytotoxic nature of NO releasing materials has been illustrated at different concentrations with a wide variety of materials such as carbosil, elasteon, silicone oil, and diatomaceous silica particle.^24,27,57,58 In addition to preventing bacterial infection, these NO releasing bone scaffolds are also expected to increase the overall healing process at the site of bone injury. Endogenous NO released by nitric oxide synthases in mammals has been shown to regulate osteoblasts activity and physiology of the bone.^59,60 Small amounts of NO released by osteoblast might act as a natural autocrine stimulator to trigger the cytokine production and osteoblast cell growth.⁶¹ In addition, research have shown that NO helps in osteogenic differentiation of bone marrow mesenchymal cells (BMSC) in 3D silk scaffolds. Nitric oxide also plays an important role in angiogenesis which would be crucial for the supply of blood at the site of bone injury.⁶² The NO release plays a critical role in tissue remodeling which would be crucial during the final phase of recovery in any musculoskeletal injury.⁶³ Studies have shown the application of NO releasing materials in tissue healing processes angiogenesis, inflammation, and proliferation.³³ The hemocompatibility, biocompatibility, and healing attributes of NO releasing polymers have also been positively proven in the recent years.^5,7,24 However, more testing on in vivo animal’s models is warranted for transitioning into clinical stages for such NO releasing biopolymeric composites.

FIGURE 8.

The WST-8 dye-based assay showing the non-cytotoxic nature of the NO releasing scaffolds on mouse fibroblast cells. The data is reported as a mean ± standard deviation for a sample size of n = 7 (for each sample type) with a p-value <0.05.

CONCLUSION

In the present study, nHA-alginate-starch and nHA-chitosan-starch scaffolds were fabricated with varying level of nHA (10–50 wt %) and tested for their load bearing capacity. About 50 wt % nHA-alginate-starch 3D scaffolds showed maximum load bearing capacity among all the scaffolds and hence were integrated with 10 wt % SNAP to further develop NO releasing antibacterial bone scaffolds. NO flux released by these 3D bone scaffolds resulted in significant eradication (>99%) of both gram-positive and gram-negative bacterial strains in addition to the excellent compressive strength. The material was shown to be non-toxic to mouse fibroblast cells. Other studies have also suggested that due to its rapid action, short half-life, and nonspecific bactericidal action, NO is unlikely to cause any resistance in the bacterial strains which is a major concern with antibiotic use. Beyond the current platform, these NO releasing 3D scaffolds can be used to achieve osteoblast growth, angiogenesis, regulation of gene expression and tissue at the site of bone injury and infections. Such NO releasing 3D bone scaffolds are not only capable of providing a 3D potential matrix to restore damaged bone function but can also reduce infections resulting in faster healing and shorter hospital stays, ultimately bringing down the cost of treatment.