Effects of Aloe Vera Gel Extract in Doped Hydroxyapatite-Coated

Jun 13, 2019 - This study aims to investigate the effects of a naturally occurring aloe vera gel extract, acemannan, in doped hydroxyapatite coatings ...
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Effects of Aloe Vera Gel Extract in Doped Hydroxyapatite-Coated Titanium Implants on in Vivo and in Vitro Biological Properties Dishary Banerjee and Susmita Bose* W. M. Keck Biomedical Materials Research Laboratory, School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164-2920, United States

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ABSTRACT: Hydroxyapatite-coated titanium alloys have been a popular choice as bone implants for load-bearing applications for the compositional similarity of hydroxyapatite to natural bone. The limited osteoinductive properties exhibited by the hydroxyapatite (HA) coatings have led to the incorporation of growth factor or dopants for improved osseointegration. This study aims to investigate the effects of a naturally occurring aloe vera gel extract, acemannan, in doped hydroxyapatite coatings on the in vitro osteoblast cell viability and in vivo new bone formation in a rat distal femur model. Silver oxide and silica-doped hydroxyapatite coatings were developed by the induction plasma spray coating method on Ti alloys to introduce antibacterial properties along with induction of angiogenic properties, respectively. The doped coating was further consecutively dip coated with acemannan to analyze its effects on the in vivo early stage osseointegration and chitosan to control the burst release of the acemannan from the calcium phosphate matrix. The results show controlled release of acemannan from the chitosan coatings, with enhanced osteoblast cell viability by the incorporation of acemannan in vitro. Improved osseointegration with a seamless implant interface and improved new bone formation was noted by the acemannan and chitosan coating in vivo, 5 weeks after implantation. Our results demonstrate the efficacy of a combination of natural medicine and naturally occurring polymer in a doped hydroxyapatite-coated titanium implant on the bone tissue regeneration for load-bearing orthopedic applications. KEYWORDS: acemannan, natural medicine, rat distal femur model, chitosan, natural polymer, bone tissue engineering

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INTRODUCTION Titanium alloys have been used widely as implant material for orthopedic surgeries for its commendable fatigue properties, biocompatibility, and low wear rates. However, due to its inherent bioinertness and lack of osteoinductive and osteoconductive properties, research was focused on the surface modification of the titanium implants.1 Hydroxyapatite (HA) coatings, owing to their compositional similarity to natural bone, were utilized to incorporate osteoconductive properties into these load-bearing titanium implants. However, the quest to incorporate osteoinductive properties into these coatings led to further research on the addition of dopants and growth factors into these coatings.2 Acemannan, an extract from the aloe vera gel, has been shown to enhance rat bone marrow stromal cell proliferation, VGEF, and alkaline phosphatase activity.3,4 Another study demonstrated higher bone mineralization with accelerated healing by the incorporation of an oral dosage of acemannan.5−7 In this study, we aim to analyze the efficacy of acemannan on in vitro osteoblast cell attachment and differentiation and in vivo early stage bone formation and osseointegration in a rat distal femur model. The prevention of primary infection in orthopedic surgeries is currently ensured by reducing the chances of contamination © XXXX American Chemical Society

and prescribing a series of antibiotics for longer terms. In the United States, only 5% of the patients undergoing orthopedic surgeries complain about primary infections. However, the severity of the consequences, including implant removal, revision surgeries, morbidity to the patient, longer recovery time than expected, and administration of another full spectrum of antibiotics, made it necessary to search an alternative to fight infections locally. Chitosan, a naturally occurring amino polysaccharide, has been demonstrated to fight against a wide diversity of microbes.8,9 This antimicrobial property of chitosan has been exploited to fabricate a diverse range of biomaterials including nanoparticles, hydrogels, and many others.8,10 Owing to its polymeric cationic structure and film-forming properties, it has been explored as a scaffold for osteogenesis and controlled release of a variety of drugs and growth factors like dexamethasone, rhBMP2.11,12,9 This study utilized a hydrophobic coating of chitosan for prevention of primary infection locally. In addition, occurrences of secondary infections are significantly higher than primary infections which again Received: January 28, 2019 Accepted: June 13, 2019 Published: June 13, 2019 A

DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

The appropriate amounts of extracted acemannan were measured out and solubilized in distilled water and mixed vigorously to ensure a homogeneous solution. Each of the Ti implants were dropcasted with the acemannan solution and dried at room temperature for at least 24 h, henceforth referred to as Ag−Si HA+acemannan. Surface Morphology by SEM. The surface of the compositions has been investigated after the drug (1 mg acemannan) and polymer (0.5 wt % chitosan) coating using a field emission scanning electron microscope (FEI Inc., OR, USA), endowed with an ETD (EverhartThornley detector), following gold coating (Technics Hummer V, San Jose, CA, USA). Acemannan Release Study. Acemannan release was measured in 0.1 M phosphate buffer (PBS) at pH 7.4 to closely imitate the physiological body conditions. Three samples with doped HA coatings, drop-casted with either 1 or 2 mg of acemannan, were placed in 4 mL of buffer solution, at 37 °C under constant shaking conditions. Buffer solutions were changed every alternate day and replaced with fresh buffer solution for the volume make up. Amounts of 0.5 and 1 wt % of hydrophobic chitosan coating in acetic acid solution were utilized for controlling the release of acemannan. The release of acemannan was measured at 540 nm after conjugation with congo red and potassium hydroxide18 using a BioTek Synergy 2 SLFPTAD microplate reader (BioTek, Wiooski, VT, USA). A standard curve was utilized to find the final cumulative percentage release of the drug. In Vitro Osteoblast Cell Material Interaction. HA and Ag−Si HA, with 1 mg of acemannan and 0.5 wt % of chitosan (optimized from effective concentration study), were seeded with 2.5 × 105 human fetal osteoblast cells (ATCC, Manassas, VA), in DMEM medium enriched with 10% FBS and penicillin and streptomycin in a sterilized environment at 34 °C under 5% CO2, following the protocols from ATCC. After 2 and 5 days of study, the cell morphology was studied under the FESEM, and the cell viability was analyzed by the MTT assay, as described before.16 In Vivo Study. Surgeries were performed on a total of 15 male Sprague−Dawley rats (Envigo, Wilmington, MA, USA), with average body weight 320−340 g, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC), Washington State University. Surgery and Implantation Procedure. The animals were anesthetized using a combination of isoflurane with oxygen. A gradual increment in drill bits up to 3 mm was used to drill a circular defect into the distal femur of the rat. Ag−Si HA implants were drop casted with 1 mg of acemannan and henceforth referred to as Ag−Si HA+Acemannan, whereas a coating of 0.5 wt % of chitosan was utilized to control the release of acemannan and reduce inflammation, referred to as Ag−Si HA+Acemannan+Chitosan. Each of these four implants (n = 6) were press fit into the drilled defect following an incomplete randomized block design.19 Following implantation, the incision was closed using a Monocryl suture.20 The animals were euthanized 5 weeks after implantation using an overdose of carbon dioxide, and the femurs were preserved in 10% neutral buffered saline. Twenty-seven of these animals were euthanized using an overdose of CO2 after 5 weeks of implantation, and the femurs were removed and preserved in 10% neutral buffered saline. Three animals implanted with doped implants were utilized for the urine collection and were euthanized using a similar procedure 9 weeks after implantation. Histomorphology. The interfacial gaps or defects at the host implant interface were analyzed after the removal of the femur from a series of radiographs acquired by an X-ray source (IVIS Spectrum CT, WSU). The series of radiographs are used to develop a 3D image of the bone by the Living Image Software. The femurs were then dehydrated in a series of ethanol and acetone and further embedded in Spurs resin. Perpendicular cross-section tissue slides of around 200 μm were cut using a low-speed diamond cutter and then mounted on positively charged slides. The slides were then stained by modified Masson Goldner trichrome staining and observed under an optical microscope. Three optical micrographs, from each implant, totaling 9 images from each sample were analyzed to quantitatively measure the bone

necessitates revision surgery, implant removal, and higher recovery time for the patients. The antimicrobial effects of silver, with demonstration of low cytotoxicity, has also been well documented.13 In this study, silver ions have been incorporated into HA coatings to prevent chances of secondary infections. However, studies revealed that a higher dosage of silver controlled secondary infections very efficiently, but it also causes cytotoxicity to the surrounding cells.14 Hence, this study intended to incorporate another biomolecule along with silver with a potential to alleviate any prospective negative effects of silver along with retaining the antimicrobial properties of silver.15 The inorganic part of the bone is made up of carbonated HA where the functional groups can be substituted by cationic or anionic groups for enhancement of osteogenic and angiogenic properties of the implants and diminish chances of secondary infections.2 Previous studies from our group have shown the importance of silicon in the development of blood vessels around the implant interface within 12 weeks of surgery in a rat distal femur model.2 Synthesis and structural stabilization of collagen by silicon intake has also been studied. Thus, the combination of Si and Ag was selected for improving osteogenic and angiogenic properties of the coatings along with controlling secondary infection. Considering much evidence from the literature, we studied the effects of acemannan and chitosan incorporation in Si4+and Ag+-doped HA-coated titanium on biological responses, in vitro and in vivo in the rat distal femur model. To the best of our knowledge, the combined effects of acemannan and chitosan in Si4+- and Ag+-doped HA-coated titanium on targeted drug delivery, in vitro osteoblast cell proliferation, and in vivo early stage bone formation have not been examined. We hypothesize that incorporation of acemannan and chitosan with cationic dopants in HA coatings will enhance osteoblast cell proliferation in vitro and improve bone bonding at the implant interface in vivo. This study reports the promotion of osteoinductivity by acemannan and Si4+ incorporation on already established osteoconductive HA coatings. Primary and secondary infection control was also enhanced by chitosan coating and the Ag+ dopant, respectively.

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MATERIALS AND METHODS

Preparation of HA Coatings. A 2 mm thick grade 5 Ti6Al4 V sheet, purchased from Titanium Joe, Canada, was sandblasted using 80 grit garnet sand in a standard sandblast cabinet and cut into discs of 12.5 mm in diameter for all release and in vitro studies. For the in vivo study, grade 5 Ti6Al4 V rods (Tiger Titanium, LV, USA) were purchased and were turned down to 2.8 mm in diameter. After ultrasonicating in water and acetone three times each, the discs and the rods were coated with HA powder (Monsanto, USA), using an inductively coupled RF plasma spray system from Tekna Plasma Systems, Canada,16 referred to as HA henceforth. After coating, the rods were cut into smaller rods 5 mm in length. HA powder doped with 0.5 wt % of silica and 2 wt % of silver was also prepared by dry mixing the powders for 2 h using 2:1 powder:milling media. The doped HA powder was then used similarly to coat another group of the Ti alloy discs and rods, referred to as Ag−Si HA henceforth. Extraction of Acemannan from Aloe Vera Gel. Organic aloe vera plant leaves of more than 3 years old (Nature Eartherapy) were decontaminated using 3% hypochlorite before the extraction process was carried out. After cutting the rind with a scalpel, the scooped-out gel was placed in a thimble. The extraction of acemannan from the aloe vera gel was done by the ethanolic Soxhlet17 extraction procedure and continued for 48 h at ∼80 °C. The acemannan powder was obtained by evaporation of the solvent at 37 °C. B

DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 1. Surface morphologies of the compositions by SEM. A more flattened surface was noted after the chitosan coating. growth around the area of implantation by ImageJ software. A region of interest of 200 μm was selected around the implant interface to access the osteoid and the total bone volume in the implant interface. Furthermore, the stained slides were observed under FESEM following gold coating to carefully observe the interface of the implant. Ion Concentration from Urine. The in vivo degradation of the implants was measured by calcium, silver, and silicon concentrations in the rat urine using inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent 7700, WSU), after removing the organic contents by acid digestion by 2% HNO3. Unknown and standard solutions were freshly prepared before measurement using stock solutions purchased from High-Purity Standards (Charleston, SC, USA). The data have been presented after taking the estimated mean from three technical and three biological replicates. Bacterial Study. A disk diffusion test was performed to assess the antibacterial properties of the acemannan, chitosan, and silver as a dopant. Similar concentrations of acemannan and chitosan (0.5 wt %) used for the in vivo were loaded into sterilized antibiotic sensitivity disks and left to dry. Bacterial cultures of Staphylococcus epidermidis (S. epidermidis) (Carolina Biological Supply Company, Burlington, NC, USA) were purchased in the form of MicroKwik Culture pathogen vials. Cells were rehydrated per company specifications and left to incubate at 37 °C for 48 h. S. epidermidis was inoculated onto nutrient agar plates. Disks were placed following inoculation and after 24 h of incubation at 37 °C, plates were analyzed for the respective zones of inhibition. Statistical Relevance. The quantitative analysis has been presented in this study as estimated mean ± standard deviation. Statistical relevance has been demonstrated by a two-way ANOVA model, and p values less than 0.05 and 0.001 have been marked as significant and extremely significant, respectively.

Figure 2. Cumulative percentage release data of acemannan in pH 7.4 measured at 540 nm by conjugation of congo red and potassium hydroxide from two different concentrations of acemannan controlled by two different concentrations of chitosan coating. The hydrophobic nature of the chitosan coating controlled the release of the hydrophilic acemannan in the physiological pH. With higher concentration of the chitosan coating, lower release of the drug was observed.

RESULTS Surface Morphology. Figure 1 shows the surface morphologies of the plasma-coated implants after the drug and polymer coating. The doped plasma-coated implants with or without acemannan showed similar surface morphology with the presence of some pores. The surface morphology was observed to be flatter with lesser pores after the lyophilization with chitosan. Acemannan Release. The cumulative percentage release of acemannan from doped HA coating with or without chitosan has been demonstrated in Figure 2. Both concentrations of acemannan showed a burst release of ∼80% within the first 72 h, after which it plateaued. The hydrophobic coating of chitosan was observed to control the release of acemannan. An amount of 1 wt % of chitosan reduced the release of acemannan from 80% to 23% over a week, where 0.5 wt % of chitosan reduced the release to 32%. Similar trends of release were noted from 2 mg of acemannan on the surfacemodified metallic substrates.

In Vitro Osteoblast Cell Material Interaction. Figure 3 demonstrates the osteoblast cell viability by MTT assay and the cell morphology by SEM, 2 and 5 days after culture. Flattened cells with well-developed filopodia were noted on HA and Ag−Si HA implants after 2 days of culture. Acemannan incorporation enhanced cell density significantly, also correlated by the cell viability assay. Chitosan coating showed a comparatively flattened surface morphology with lesser pores and showed no significant difference in cell viability compared to control HA coating. A lot of apatite formation was observed on the surfaces of the implants after 5 days of incubation. Excellent cell morphologies were noted on all surfaces with similar trends in cell viability at day 5. In Vivo Cell Material interaction. Histological Evaluation and CT Scan Analysis. The interface of the implant 5 weeks after implantation was analyzed by CT scan radiographs, presented in Figure 4. Proper bone lodging was observed in all the implants from the low-resolution CT scan micrographs, without much significant difference among the different compositions. Notable gaps had been noted at the implant

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DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 3. In vitro osteoblast cell morphology and cell viability of HA-coated Ti64 implants with or without dopants and acemannan as observed 2 and 5 days after incubation. Excellent cell attachment on the acemannan-coated samples reveals the influence of acemannan on osteoblast cell proliferation, also consistent with the quantitative cell viability measurements by MTT assay. The silver and silicon dopants along with chitosan at all time points show no significant difference in cell growth compared to the control HA coatings (** presents p < 0.001, extremely significant).

interface in the HA and Ag−Si HA implants. No distinct interface of the implant had been demonstrated in the acemannan- and chitosan-coated doped implants. Modified Masson Goldner trichrome staining was used to analyze the effects of acemannan and chitosan on new bone formation at the host implant interface. Bluish green presents mineralized bone, whereas the reddish orange color shows the osteoid formation. The optical micrographs demonstrated reddish orange color at the interface of the implant with the incorporation of acemannan, with no visible gaps, revealing enhanced new bone formation. Chitosan coating demonstrates

improved bluish green color at the implant interface, revealing mineralized bone and hence accelerated healing. Histomorphometric analysis, performed by calculating the color density around the region of interest using ImageJ, has been presented by Figure 5. Enhanced osteoid formation had been observed by acemannan incorporation. Lower osteoid around the implant interface with high total bone volume reveals enhanced mineralization with the chitosan coating. A closer look at the interface observed by FESEM demonstrated notable gaps in the HA and Ag−Si HA coatings. A seamless D

DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 4. CT scan (a) histology and SEM micrographs (b) of HA-coated Ti64 implants with or without dopants and acemannan. The presence of acemannan improved the osteoid formation which is evident from the enhanced reddish orange color around the vicinity of the implant. SEM micrographs also showed no gaps at the interface of the acemannan implants. The combined presence of chitosan with acemannan further mineralized the new osteoid formation around the implant, shown by the greenish blue color, leading to improved early stage osseointegration. Osseous tissue had been observed to interlock with the implant in the SEM micrographs, leaving no visible gaps.

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interface on the other hand had been noted with acemannan and chitosan incorporation. Ion Concentration in Urine. The degradation of the coating from the implants was analyzed by measuring the Ca2+, Si4+, and Ag+ ions in the rat urine of the animal which received the doped HA-coated implant. Figure 6 shows the ion concentrations in the urine of the animals up to 60 days post implantation. Higher concentration of calcium ion, observed in the urine of the rats, was expected compared to the amount of the dopants. Demonstration of Bacterial Efficacy. Zones of inhibition had been demonstrated 18 hrs after inoculation in Figure 7. The acemannan coating of 1 mg/ml had demonstrated a clear zone of inhibition in the nutrient agar plate and the combination of acemannan with chitosan presented a larger zone of inhibition compared to only acemannan incorporation. This reveals the anti-bacterial effects of the natural medicinal compounds along with the natural polymer, leading to accelerated healing.

DISCUSSION

Acemannan, a natural medicinal compound, has been used for decades in Asian countries for wound healing and has received prominence in the United States after successful treatments of skin burns caused by radiographs.21 Further research showed the importance of acemannan in the treatment of bone disorders5.6 This study aims to analyze the effects of acemannan, extracted from aloe vera and chitosan in doped hydroxyapatite coatings on biological response. Figure S1 demonstrated the characteristic peaks of acemannan in both NMR and FTIR spectra. Hydroxyapatite has been utilized as a coating for metallic load-bearing implants since the 1980s owing to its compositional similarity to natural bone and adequate surface chemistry to support new bone formation.22 The coatings currently are being employed as an alternative to cemented implants for accelerated healing in younger patients and reduced chances of revision surgeries. The presence of trace elements like silicon and silver augment the osteoconductivity of HA with osteoinductivity and antibacterial properties.2,15 E

DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 5. Percentage of osteoid formation and total bone area around the region of interest (200 μms) of the implant, estimated using the ImageJ software. No significant difference in osteoid formation has been noted around the doped HA coating and the acemannan implants; however, higher bone mineralization revealed higher total bone volume. Lower osteoid formation along with higher bone surface area in the presence of chitosan demonstrate enhanced mineralization 5 weeks after implantation, leading to accelerated healing.

Figure 6. Degradation of the HA coating from the titanium alloy measured by the ion concentration of the rat urine. The release of the ions from the doped HA coating reveals the presence of the coating in vivo 9 weeks after implantation. Cumulative release of silver was deemed effective in controlling the growth of bacterial strains, along with being nontoxic to humans.

of scar or granular tissue24 and has also been observed to be effective against a wide variety of bacterial infections.25 In this study, we have utilized deacetylated chitosan in controlling primary infection and preventing inflammation, leading to accelerated healing. Diseases like osteomyelitis caused by bacterial infections occur even weeks to months postsurgery.26,14 S. epidermidis, S. aureus, and E. coli are some of the most common pathogens

Postsurgical infections at the site of implantation or at the surgery site remain a concern for load-bearing implants. A spectra of postsurgical antibiotics or other antimicrobial remedies have not witnessed much success over the years, leading to the quest for finding other treatments. Chitin and chitosan, hydrophobic amino polysaccharides, have been popularly used for accelerated wound healing in humans.2313.10 Chitosan has been shown to heal tissues without any formation F

DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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The effects of acemannan and chitosan on in vitro osteoblast cell material interaction have been demonstrated in Figure 3 for a span of 5 days. Flattened cells with extended filopodia on all compositions reveal biocompatibility of the coatings. Statistically significant improvement in cell viability with acemannan incorporation proves the efficacy of acemannan on enhanced osteoblast cell proliferation. The qualitative SEM images also correlate to the quantitative cell viability by MTT assay. Chitosan incorporation did not significantly alter the osteoblast cell viability over the span of 5 days, owing to the interference of chitosan with the cell membrane.33 Figure 4 demonstrates the in vivo new bone formation in the rat distal femur model 5 weeks after implantation. 3D images developed by CT scan radiographs in Figure 4a reveal proper bone lodging in all the implants. Notable gaps are observed at the implant interface of HA and Ag−Si HA implants, whereas a seamless interface between the host bone and the implant has been observed by the acemannan and chitosan coating. The initial recruitment of the preosteoblasts and their differentiation into mature osteoblasts determines the healing of the bone. Metallic implants require 3−4 weeks for the healing procedure to initiate.34 In this study, we investigated the effects of the acemannan and chitosan in the acceleration of the new bone formation, right after the initiation of a healing procedure by the optical micrographs after Masson Goldner staining (Figure 4b). Increased osteoid formation by enhanced reddish orange color around the implant interface was seen by the acemannan coating compared to HA, consistent with the histomorphometric analysis shown in Figure 5.3 Enhanced early stage osteogenesis is hence witnessed with the incorporation of a natural medicinal compound into the doped HA coating without the presence of growth factor or steroids.16 The incorporation of chitosan on the other hand shows improved total bone volume, with lower new bone formation. The bluish green color around the implant reveals mineralized bone as early as 5 weeks after implantation. Although histomorphometric analysis shows comparable osteoid formation with the presence of the dopants with or without acemannan, optical micrographs clearly reveal a gap around the implant. To get a better analysis of the osseointegration, FESEM was utilized to observe the implant interface. Notable gaps around the implant in the HA coatings with or without dopants reveal no to lower osseointegration. The seamless interface with the acemannan and chitosan coating, also revealed from the CT scan, proves the efficacy of the natural medicinal compound and natural polymer on in vivo early osseointegration. The degradation of the HA coating was investigated from the ion concentrations in the urine of the animal, demonstrated in Figure 6. The measure of almost similar concentrations of the calcium ion in the urine of the rat suggests gradual degradation of the coating and reveals the presence of the coating in the animal body 9 weeks after implantation. Also measured is the silver ion concentration in the rat urine. The reported minimum inhibitory concentration of silver for the prevention of the growth of a wide spectrum of bacterial strains including S. epidermidis, S aureus, and E. coli ranges from 0.2 μg to 20 μg per mL.35 However, the minimum inhibitory concentration of silver deemed nontoxic in the human body and effective against the bacterial strains is below 10 μg per mL or 10 ppm.35−37 The cumulative measured silver concentration excreted by the rat was lower than 10 ppm after 9 weeks of implantation. The efficacy of the natural medicinal

Figure 7. The zone of inhibition is demonstrated by the inoculation of S. epidermidis for 18 hours. Presence of acemannan shows a clear zone around the disc, whereas a larger clear zone observed around the acemannan and chitosan coating present the anti-bacterial effect of the acemannan and the combination respectively.

causing bacterial infection in medicinal implants. Antibiotics have not been witnessed to be effective in treatment of these secondary infections. Much solace was found in the possibility of the treatment of secondary infection by exploiting the antimicrobial effects of silver.27,28 The affinity of silver toward the sulfur, nitrogen, or oxygen forming silver salts29 along with its interaction with the thiol and the amino groups30 interrupt many biochemical pathways, resulting in the prevention of microbial growth. Although biocidal in nature, treatment of primary infections has been performed by appropriate doses of silver coating. In this study, 2 wt % of Ag has been utilized as a dopant into HA coatings for load-bearing implants to control primary infection. Figure 2 shows the release profile of acemannan investigated from two different concentrations of chitosan coating. Burst release of biomolecules is very inherent to calcium phosphatebased delivery systems.31 A burst release of hydrophilic acemannan of ∼80% was noted within 72 h without any polymer coating. The release of a biomolecule in vivo is controlled by the matrix degradation or diffusion and chemical mechanisms, as seen from Figure 1. Diffusion is a faster process compared to the matrix degradation, so the release of acemannan over the 2 weeks has been noted to be diffusion dominated, owing the hydrogen bonding with the aqueous environment.31 However, the burst release of acemannan from the doped HA coating does not serve the purpose of local drug delivery for effective treatments. Both chemistry and surface topography of the doped plasma coating and the hydrophilic acemannan coating affect the wettability, and a decrease has been noted in contact angle from ∼18° for HA coating to ∼5° in the presence of the hydrophilic drug, acemannan. The hydrophobicity of the chitosan coating controls and sustains the release of acemannan in the physiological pH. An amount of 0.5 wt % of chitosan led to a cumulative release of 32% of 1 mg of acemannan compared to 80% over a week, whereas 1 wt % coating led to a reduced release of 23%. Very similar release profiles have been noted from 2 mg of acemannan release. The favorable hydrogen bonding of chitosan with acemannan in the physiological pH controls the release of the biomolecule.32 G

DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Valerie Lynch-Holm and Dan Mullendore from Franceschi Microscopy & Imaging Center, WSU, for their assistance with microscopic techniques.

compounds on infection control has been shown in Figure 7. The zones of inhibition for Gram-positive bacteria S. epidermidis have been observed to be larger for the combination of acemannan (1 mg/mL) and chitosan coating (0.5 wt %) compared to only acemannan coating. The results of this study show the combined influence of the acemannan and chitosan on doped HA coatings for loadbearing applications. These implants have been shown to have the potential to be used as a next-generation biomaterial with natural medicine, with improved early stage osseointegration and accelerated healing.

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CONCLUSIONS Acemannan, a natural medicinal compound, extracted from aloe vera gel has been investigated for its effects on in vitro and early stage in vivo biological response for load-bearing applications. Silver has been used as a dopant in the hydroxyapatite coatings on titanium alloy for the prevention of secondary infections and to reduce chances of revision surgeries for younger patients, whereas the silicon is explored due to its angiogenic efficacy. The burst release of acemannan from the hydroxyapatite coating is controlled by using a coating of a hydrophobic natural polymer, chitosan. The chitosan coating has also been studied for its antibacterial efficacy, demonstrated by the zone of inhibition of S. epidermidis, 18 h after inoculation. Improved osteoid formation from 34% in control HA to ∼49% with the incorporation of acemannan in doped coatings reveals the efficacy of acemannan on early stage osteogenesis, 5 weeks after implantation in the rat distal femur model. The seamless interface observed by FESEM micrographs also shows early stage osseointegration in vivo. Chitosan coating with the combination of acemannan witnesses higher bone mineralization and enhanced osseointegration, leading to accelerated healing. The results from this study prove the potencies of the combination of a natural medicinal compound with a natural polymer on doped hydroxyapatite coating for accelerated healing in load-bearing orthopedic applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00077. Characterization of the extraction of acemannan by NMR and FTIR, showing the characteristic peaks of acemannan (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Dishary Banerjee: 0000-0001-6682-9258 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge financial assistance from the National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS, NIH) under Grant Number R01AR-066361. They would also like to thank Samuel Ford Robertson with his help for the plasma sample preparation and H

DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsabm.9b00077 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX