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. Author manuscript; available in PMC: 2021 Jul 25.
Published in final edited form as: Surf Coat Technol. 2020 Apr 15;394:125793. doi: 10.1016/j.surfcoat.2020.125793

Effects of vitamin C on osteoblast proliferation and osteosarcoma inhibition using plasma coated hydroxyapatite on titanium implants

Naboneeta Sarkar 1, Hailey Morton 1, Susmita Bose 1,*
PMCID: PMC7328859  NIHMSID: NIHMS1591470  PMID: 32612317

Abstract

Plasma-sprayed hydroxyapatite (HAp) coated titanium (Ti) implants are being extensively used in orthopedic surgeries and post-tumor resection to repair load-bearing segmental bone defects. In this study, vitamin C, an abundantly available natural biomolecule, is loaded onto plasma-sprayed HAp-coated commercially pure titanium (cpTi) surface to evaluate its chemopreventive and osteogenic properties, suggesting its clinical significance as an alternative or adjunct therapy in the treatment for osteosarcoma bone resection. Controlled release of vitamin C from HAp coated cpTi implant is assessed by in vitro drug release study, where Korsmeyer-Peppas model was applied to understand the release kinetics. After 21 days, the implants loaded with 400 and 800 μg of vitamin C showed a cumulative release of 62.7 and 74.1% in acidic microenvironment, whereas, 50.9% and 53.1% of total vitamin C release were observed by the implants loaded with 400 and 800 μg of vitamin C in physiological pH, respectively. To observe the effects of in vitro vitamin C release on osteosarcoma and osteoblast cellular activity, MG-63 (human osteosarcoma) and hFOB (human fetal osteoblast) cells were cultured on the surface of the implant and MTT cell viability assay and FESEM were carried out at 3 and 7 days of culture. Presence of high dosages 25 mM vitamin C shows a statistically significant (p≤0.05) decrease in osteosarcoma cell viability after 3 days, while both 5 mM and 25mM vitamin C reduced cellular viability by 2.5 folds (p≤0.05) compared to the control after 7 days. Interestingly, the presence of vitamin C showed no obvious signs of cytotoxicity towards osteoblast cell-line at day 3 and day 7, as confirmed by the MTT assay. Additionally, the FESEM images depict layers of hFOB cellular morphology on the surface of the implants, suggesting excellent cytocompatibility towards the osteoblast cells. These results suggest that vitamin C loaded HAp coated cpTi implant with improved osteogenic and chemopreventive properties can be considered as a promising reconstructive option to repair the post-tumor resection defects in osteosarcoma.

Keywords: In vitro vitamin C release, Plasma coated HAp, load-bearing Ti implant, Osteoblast cell culture, Osteosarcoma cell culture

Graphical Abstract

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1. Introduction

Vitamin C is one of the most popular alternative medicines, has been used orally or intravenously for almost 70 years. It has been used to treat common cold, infections, diabetes, atherosclerosis and various autoimmune diseases [1]. Its chemopreventive property has been highly debated since Pauling promoted vitamin C as an anti-cancer agent in 1976 [2]. Studies showed high plasma concentration of vitamin C (1 mol/L) has selective toxicity towards tumor cells but not to the normal cells [3]. In contrast, 10 grams of daily oral dosage showed no pharmacological benefit to cancer patients because the maximum tolerated oral dose cannot reach the effective plasma concentration [4]. Some significant clinical evidence showed cytotoxic effects of vitamin C on some cancer cells, however, the mechanism was not fully elucidated [5–6]. One of the possible mechanisms was attributed to its antioxidant property, where it acts as “free radical scavengers” and thus reduces cancer-causing oxidative stress [7].

In view of the aforementioned, we were interested in investigating the effect of vitamin C on MG-63 bone cancer cells. Unlike other forms of cancer, osteosarcoma is mainly diagnosed in adolescents and young adults [8] and is considered the most prevalent primary bone malignancy [9]. Metastatic bone cancer has a very high incidence rate since the bone tissue is one of the most preferred sites for metastases of any malignant tumor [10]. Treatments of bone tumors involve chemo-, radiotherapy, and surgical resection. However, the drugs used for chemotherapy exhibit several significant side effects due to their toxicity towards the normal tissues, while bone tumor cells are generally radio-resistant [11–12]. Surgical resection of the tumor and surrounding bone tissue does not eliminate the cancer cells completely and also creates bone defects, requiring bone graft implantations. Therefore, fabrication of a bifunctional implant seems to be a straightforward approach for the treatment of malignant bone tumors, which repairs bone defects by bone regeneration and simultaneously release chemopreventive agent that eliminates the residual bone tumor cells without causing serious side effects. To promote faster bone ingrowth and regeneration in the defect sites, various antibiotics, growth factors, and biomolecules are often introduced to influence the biological performance of the implant.

Metallic biomaterials, especially titanium (Ti) and its alloys are considered suitable candidates for load-bearing hip or knee implants due to their optimal mechanical properties, such as high toughness and fatigue resistance [13]. The surface of the Ti implant is often modified with calcium phosphate (CaP) based ceramic coating to promote proper biological fixation by preventing fibrous tissue encapsulation after implantation [14–15]. Synthetic hydroxyapatite (HAp), a CaP ceramic, is compositionally similar to the inorganic component of bone. Along with excellent biocompatibility and bioactivity, HAp also exhibits osteoconductivity, which allows better proliferation, migration, and attachment of bone cells [16–17]. Among various coating techniques, plasma spray continued to be the only FDA approved process and it is preferred over other coating methods due to its simplicity, high deposition rates, low temperatures applied to the substrate, and cost efficiency [18–20].

In this work, HAp was coated on the cpTi substrate by plasma spraying process, and vitamin C as an osteoregulator and chemopreventive agent was incorporated on the coated load-bearing implant. The effect of pH and drug concentration on in vitro vitamin C release study was assessed. In addition, in vitro osteoblast and osteosarcoma cell culture were carried out. We hypothesized that vitamin C incorporated HAp coated cpTi implant would (i) exhibit controlled in vitro release kinetics in both physiological and acidic pH conditions (ii) inhibit the proliferation of osteosarcoma (MG-63) cells in a dose-dependent manner, and (iii) enhance the proliferation of healthy osteoblast (hFOB) cells. Schematic illustration of the processing of plasma coated HAp on cpTi surface and a brief mechanism of action of vitamin C on inhibition of tumor cells and proliferation of osteoblast cells is shown in Fig. 1.

Fig. 1.

Fig. 1

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Clinical significance of plasma coating and in vitro release of vitamin C from plasma coated cpTi implant.

2. Materials and methods

2.1. In vitro release of vitamin C

2.1.1. Preparation of coating

Titanium plates, purchased from President Titanium (MA, USA), were cut by water jet cutter into discs with a diameter of 1.22 cm and thickness of about 2 mm. Subsequently, these discs were sandblasted, cleaned ultrasonically in DI water and treated with acetone to ensure the remaining organic material was removed. HAp powder (Monsanto, MO, USA) were sieved to receive 150–212 μm particle size prior to coating. Afterward, coatings were applied using a 30 kW inductively coupled radio frequency (RF) plasma spray system (Tekna Plasma Systems, Canada). The system is equipped with a supersonic plasma nozzle and an axial powder feeding system. 25 kW plate power and 110 mm working distance were maintained for this work. Argon (Ar) gas was used as the central and carrier gas with a flow rate of 25 and 13 standard liters per minute (slpm). A mixture of 60 slpm argon and 6 slpm hydrogen were used for sheath gas. Chamber pressure was kept at 5 pound-force per square inch gauge (psig).

2.1.2. Vitamin C loading and release study

For the in vitro release study, vitamin C (Sigma Aldrich, MO) was dissolved in DI water at a concentration of 4 and 8 mg/ml. 100 μl of total drug solution was loaded on both surfaces of HAp coated cpTi disc, using a pipette. This concentration corresponds to 400 and 800 μg of vitamin C on each implant. In vitro release study was performed at 0.1 M pH 7.4 phosphate buffered saline and pH 5.0 acetate buffer (n=3). These pH levels are used to imitate the physiological pH and the acidic environment right after surgery, respectively. A pH probe was used to ensure the measured pH was within ±0.05 range. Subsequently, implants immersed in 4 ml of buffer solution were placed in an orbital shaker at 150 rpm at 37°C. At each time point, buffer media was collected and then refilled with the respective pH buffer. Buffer media was collected after selected time points; 1.5, 3, 6, 12, 24 hours and then 2, 3, 5, 7, 10, 14, 18, and 21 days. The concentration of vitamin C released at selected time points was measured using a Biotek Synergy 2 SLFPTAD microplate reader (Bioteck, Winooski, VT, USA). Briefly, 200 μl of collected media was pipetted in a 96-well plate and the absorbances of each well were measured at 265 nm wavelength. Finally, drug concentration was measured using a standard curve and plotted against time.

2.2. In vitro cell culture study

2.2.1. Osteosarcoma cell culture

Prior to cell culture, vitamin C dissolved in aqueous solution was pipetted on top of the HAp coating so that the drug concentration reaches 5 and 25 mM vitamin C per implant. For control, HAp coated cpTi implants were used without any treatment. All implants were autoclaved at a temperature of 121 °C for 60 minutes. MG-63 (Human osteosarcoma cell line) used in this study were purchased from ATCC, USA. To maintain the culture, eagles minimum essential medium (EMEM) (ATCC, USA) was used, as suggested by the company. The cell media was changed every 2 days. After the cells reach confluency, implants were kept in 24 well plates and cells were seeded onto the implants at a density of 2×106 cells/mL. The incubator temperature was kept at 37°C and a 5% CO2 atmosphere was maintained for the entire study.

2.2.2. Osteoblast cell culture

hFOB (human fetal osteoblast) cell line purchased from PromoCell GmbH, Germany, were used in this study. Cells were grown in a culture flask using osteoblast growth medium (PromoCell GmbH, Germany). The growth medium was changed every 2–3 days. The cells were kept in an incubator maintained at 37°C under 5% CO2 atmosphere, as recommended by the company. The sterilized implants were placed in 24 well plates and confluent hFOB cells were seeded at a density of 2×106 cells/mL on top of the implants.

2.2.3. Cell morphology

Samples for FESEM (Field Emission Scanning Electron Microscope) were taken out at 3 and 7 days to characterize cellular morphology. 1 ml of 2% paraformaldehyde/2% glutaraldehyde in 0.1M phosphate buffer was added to fix the implants. Fixation is carried out to preserve the implant to its natural state during the process of characterization. After fixation, the implants are kept at 4 °C overnight. Afterward, post-fixation was carried out for 2 hours using 2% osmium tetroxide (OsO4) at room temperature. Thereafter, ethanol dehydration (30%, 50%, 70%, 95%, and 100% three times) was performed, followed by hexamethyldisilane (HMDS) drying. A gold sputter coater was used to coat the implants. The morphology of implants was then studied using FESEM (FEI 200F, FEI Inc., OR, USA).

2.2.4. Quantitative analysis

hFOB and MG-63 cell viability were evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, St. Louis, MO) assay. To prepare the MTT solution, 5 mg of MTT is dissolved in 1 ml of sterile-filtered PBS. 100 μl of MTT solution was then added to each sample in 24-well plates followed by the addition of 900 μl of cell medium. The samples were incubated for 2 h. 1 ml of MTT solubilizer is prepared using 10% Triton X-100, 0.1N HCl and isopropanol. After incubation, 600 μl of MTT solubilizer was added to dissolve the formazan crystals. Thereafter, 100 μl of that solution was transferred into a 96-well plate and read by UV–Vis spectroscopy microplate reader (BioTek) at 570 nm. To ensure reproducibility, all samples were used in triplicate. Data are presented as mean ± standard deviation. Student’s t-test was used to perform statistical analyses and P values < 0.05 and <0.0001 are considered significant and extremely significant.

3. Results

3.1. HAp plasma coated cpTi implant and in vitro vitamin C release kinetics

Fig. 2 shows the surface morphology of bare cpTi disc and plasma coated HAp surface. The HAp coated microstructure shows crack-free, granular morphology with the presence of micropores.

Fig. 2.

Fig. 2

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Surface morphology of the (a) bare cpTi disc and (b) plasma sprayed HA-coated cpTi (before drug release) showing porous HA coating on Ti disc.

Fig. 3 shows in vitro release kinetics of vitamin C from HAp coated cpTi implant at pH 7.4 and pH 5.0. The controlled release of vitamin C has been reported for 21 days and the release amounts were dose-dependent. Lower drug release amounts were found at pH 7.4 as compared to acidic pH. After 21 days of release, 74.1% of the total drug was released from the implants loaded with 800 μg of vitamin C, whereas 62.7 % drug was released from the implants loaded with 400μg of vitamin C. Release amounts were higher in acidic solution than in physiological pH and can be attributed to the greater rate of dissolution. After 21 days, 53.1% of total release amounts were achieved for the implants loaded with 800 μg and 50.9% for the ones loaded with 400 μg of vitamin C.

Fig. 3.

Fig. 3

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In vitro release kinetics of vitamin C at pH 5.0 and pH 7.4 fitted in Korsmeyer-Peppas model. Release kinetics of vitamin C loaded samples at pH 7.4 showing only 6.84% and 13.09% release of drug for 21 days from samples containing 800 and 400μg of vitamin C, respectively. (top left) Release kinetics of vitamin C loaded samples at pH 5.0 showing 9.56% and 16.46% release of drug for 21 days from samples containing 800 and 400μg of vitamin C, respectively.

Surface morphology of vitamin C loaded implants at pH 7.4 and 5.0 after 21 days is shown in fig. 4. The degradation of surface, cracks and exposed pores, can be attributed to surface degradation caused by the acidic release media and in turn, affected the overall drug release kinetics in pH 5.0. Comparatively, much lesser surface degradation can be seen at pH 7.4, which resulted in a slower release of vitamin C in physiological pH.

Fig. 4.

Fig. 4

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FESEM images of vitamin C loaded samples at pH 5.0 and 7.4 after 21 days of release showing formation of large cracks and pores in samples present in acidic pH (pH 5.0) compared to the physiological pH (pH 7.4).

3.2. Effects of vitamin C on in vitro osteosarcoma and osteoblast cell proliferation

To investigate the effect of vitamin C on osteosarcoma cell viability, MTT assay was carried out at 3 and 7 days of cell culture. Quantitative analysis in Fig. 5(a) shows the presence of vitamin C has a pronounced effect on the inhibition of osteosarcoma cell proliferation. At day 3, the implants containing 5mM vitamin C were not able to exert any chemopreventive effect on osteosarcoma, however, the presence of 25mM vitamin C showed a statistically significant reduction in cell viability (p≤0.05). At day 7, 5mM and 25mM vitamin C have significantly decreased osteosarcoma cell viability by almost 2.5 folds (p≤0.05). Besides, while control significantly increased osteosarcoma cell viability from day 3 to day 7 (p≤0.05), the presence of vitamin C arrested the osteosarcoma cell proliferation at day 7 by showing almost no statistical increment in cellular viability from day 3. FESEM images in Fig. 5(b) shows osteosarcoma (MG-63) cellular morphology on control: HAp coated cpTi implants; and 5mM/25mM vitamin C loaded on HAp coated cpTi implants surfaces at day 3 and 7, respectively. At day 3, layers of osteosarcoma cells with firm adhesion and filopodial attachment can be seen in the control implant without vitamin C. Similar or little lower osteosarcoma cell attachment and proliferation are exhibited with 5mM of vitamin C implants. However, a significant inhibition in osteosarcoma cell proliferation is shown in implants loaded with 25mM vitamin C, at day 3. Subsequently, at day 7, we can see a multilayer formation of osteosarcoma cells in implants containing no vitamin C, which is in accordance with the MTT assay. Similar to day 3, implants loaded with 5mM vitamin C showed no significant reduction of osteosarcoma cell proliferation. However, a much lesser amount of osteosarcoma cell coverage can be seen on the surface of the implants with 25mM vitamin C, which supports our hypothesis that vitamin C is effective on osteosarcoma cell line in a dose-dependent manner.

Fig. 5.

Fig. 5

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(a) MTT osteosarcoma cell viability assay showing presence of vitamin C has significantly decreased osteosarcoma cell viability by almost 1.5 folds at day 7. (b) SEM images of osteosarcoma cell culture on vitamin C loaded HAp coated cpTi samples at day 3 and day 7 showing significantly reduced osteosarcoma cell proliferation in samples with 25 mM vitamin C.

Fig. 6(a) demonstrates the MTT osteoblast cell viability assay for HA-coated Ti, 5 and 25 mM vitamin C loaded on HA-coated cpTi implants at day 3 and day 7 of culture. Data reveals that the presence of vitamin C did not show any significant decrease in cellular viability at day 3 and day 7, compared to control. This suggests that vitamin C loaded sample shows excellent cytocompatibility with no cytotoxicity towards osteoblast cells. Fig. 6(b) shows FESEM cellular morphology for osteoblast (hFOB) cell culture on control, 5 and 25mM vitamin C loaded HAp coated cpTi implants surface at day 3 and day 7. The presence of osteoblast cells with firm filopodial attachment can be seen in control implants suggesting excellent biocompatibility of HAp coated cpTi implants towards the healthy bone cells. Regardless of concentration, the presence of vitamin C shows excellent signs of osteoblast cell proliferation, attachment, and growth on the sample surface. Besides, layer-like osteoblast cell spreading can be seen at day 7 in the presence of 25mM vitamin C. The presence of filopodial extensions and no obvious cytotoxicity on the implant surface suggests vitamin C does not have any adverse effect on healthy osteoblast cellular activity.

Fig. 6.

Fig. 6

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(a) MTT osteoblast cell viability assay demonstrating presence of vitamin C showed no signs of cytotoxicity against healthy osteoblast cells at day 3 and day 7. (b) SEM images of osteoblast cell culture on control and vitamin C loaded HAp coated cpTi samples showing firm filopodial processes and osteoblast proliferation at day 3 and day 7.

Discussion

Complete eradication of residual malignant cells and simultaneous tissue regeneration after tumor resection is still a challenge in the area of bone cancer treatment. In the past few years, utilization of metallic Ti-based bone implants has revolutionized the conventional concepts of bone defect reconstruction, particularly those in the cases of surgical resection of osteosarcoma [21–22]. A recent study has reported a successful reconstruction of a massive bone defect caused by surgical resection of bone malignancies with the use of titanium implant, which later in a follow-up study exhibited promising osseointegration without any signs of local recurrence [22]. Currently, localized delivery of potent biomolecules from these implants has become an emerging trend to enhance functionality by promoting bioactivity, osseointegration, and suppressing the risk of bacteria-related infection. In one such study, a two-phase release profile (burst release followed by zero-order kinetics) for gentamicin was achieved from a designed bone-fixating device made of titanium wire with titania nanotube arrays [23]. Aside from preventing infection, orthopedic implants are also being developed as a release platform for growth factor and bone morphogenic proteins to promote tissue regeneration at the injury site [24]. However, at present, there is limited research on the design of Ti-based implants with controlled delivery of chemopreventive agents, which can prevent local recurrence of osteosarcoma. A recent study has presented novel chitosan- 18β- glycyrrhetinic acid-modified titanium implants with nanorod arrays, which suppressed in vitro osteosarcoma growth and promoted osteoblasts adhesion and proliferation [25]. Curcumin, a widely researched natural biomolecule has been utilized for controlled delivery from a titanium-based implant to prevent tumor recurrence and simultaneously repair surgical bone defects [26]. However, a deeper understanding of the complexities behind the biomaterials design and related drug release mechanism is necessary to achieve better control over biomolecule delivery, which can further ensure improved clinical success of such systems.

In this study, a bifunctional scaffold based on vitamin C loaded HAp-coated cpTi implant was designed and investigated for this purpose. HAp coating has been used for over a decade to improve bioactivity and osseointegration in orthopedic metal implants. Through the use of plasma-spray, HAp coatings obtain high mechanical strength but undergo some phase and crystallinity changes due to high processing temperature causing thermal decomposition and melting of particles. The HAp coated cpTi implants utilized in this study show interfacial bond strength in the range of 17.4±2.9 MPa (n=5). Besides enhancing bioactivity, HAp coating also plays an important role as a potential drug delivery carrier for localized delivery. Various anticancer drugs, bisphosphonates and growth factors such as methotrexate, zoledronate, BMP2 are often incorporated in HAp coating to prevent tumor regrowth, reduce bone resorption, and promote accelerated healing of bone defects [27–28]. Local delivery of these drugs eliminates the risk of side effects in non-target tissues by releasing the drug at the target site. Controlled drug release from thin bioactive HAp coating is often desired, to introduce osteoinductive, chemopreventive or antibacterial potential. However, in most cases, an unwanted burst release is observed, followed by a very low sustained release of the drug, which not only causes severe toxicity and tissue irritation but also creates a major disadvantage for orthopedic implants where constant release kinetics are desired for the duration of new bone formation.

In this study, vitamin C biomolecule is loaded on the HAp coating and a controlled in vitro release from the implant was monitored for 3 weeks. The cumulative release kinetics are fitted with the Korsmeyer-Peppas model, which is a characteristic model for cylindrical drug delivery systems. The in vitro release data shows that the implants in physiological buffer media exhibited a lower amount of vitamin C release compared to the acidic media due to the lack of dissolution of HAp coated particles. The implant in pH 7.4 also displays a much smaller initial burst when compared to implants in acidic solution. This smaller burst can be attributed to the fraction of the drug which is weakly bound to the implant surface. This characteristic drug release is desirable because burst release of drug at the initial time points assists in removing the residual tumor cell at acidic microenvironment, and when the tissue returns to the normal pH, the sustained release of osteogenic drug at physiological microenvironment helps to improve osseointegration and promote faster wound healing [29].

Unlike the implants immersed in the physiological buffer system, those kept at acidic pH 5.0 experience a higher degradation and particle leaching, as shown by the FESEM images taken after 21 days of drug release. The results reveal that the HAp coatings are relatively insoluble at physiological pH of 7.4, however, in acidic environments, HAp has increased solubility, which can be confirmed by the inhomogeneous surface. These phenomena are well supported by earlier reports that states, environmental factors such as the composition and pH of the buffer media system greatly affect the dissolution rate of the coatings and therefore alter the drug release [30]. It is worth mentioning that the dissolution of HAp coatings and related drug release can be also controlled by multiple factors such as the degree of crystallinity, the presence of secondary phases and porous coating [31].

Effect of vitamin C on cancer treatment has remained a subject of controversy for a long time until some recent significant findings have revived the interest in vitamin C by repeatedly demonstrating its dose-dependent toxicity against various forms of malignancies [32–34]. Studies have shown that vitamin C, when administered intravenously, selectively kills numerous cancers in xenograft models including ovarian, pancreatic, hepatocellular and glioblastoma tumors without adversely affecting normal cells. Although, quite a few in vitro and in vivo studies have demonstrated the potential of vitamin C induced cytotoxicity, the gradual release of vitamin C from an orthopedic implant and its local interaction with MG-63 osteosarcoma and normal osteoblast cells were yet to be explored. Keeping this in mind, the objective of this study was to incorporate vitamin C on bone like HAp coated implant and understand its’ activity towards both osteosarcoma and osteoblast cells.

The results from this study showed the presence of 25 mM vitamin C suppressed in vitro osteosarcoma cell viability by 2.5 folds after 7 days. This provides a ground for further investigation of high dosage of vitamin C as an alternative or adjunct treatment in osteosarcoma. The mechanism behind vitamin C-induced selective cytotoxicity has been elucidated in a recent study [35]. As per their report, the oxidized form of vitamin C or dehydroascorbate (DHA) is taken up by the cells via glucose transporters, which is also upregulated in tumor cells compared to normal cells. Therefore, tumor cells automatically uptake more glucose along with DHA compared to normal cells. Inside the cell, DHA is again reduced back to vitamin C, where it gets accumulated and acts as a pro-oxidant that produces oxidative stress. Generation of free radicals or reactive oxygen species (ROS) and hydrogen peroxide (H2O2) causes cellular damage, suppresses tumor growth and subsequently results in vitamin C-mediated cell death [36].

To ensure the clinical success of vitamin C as an adjunct therapy for bone cancer treatment, its effect on healthy bone-forming cells further needed to be investigated. The results show increased osteoblast proliferation and viability with the presence of vitamin C compared to the control. Layers of osteoblast cell morphology can be seen at day 11 with the presence of 25 mM of vitamin C. This is in accordance with the previous reports demonstrated that vitamin C is well known for enhancing collagen synthesis, the main organic constituent of bone matrix. It is an essential cofactor for the hydroxylation of proline and lysine residues in collagen and therefore it helps to maintain collagen structure [37]. A recent study also documented that vitamin C enhanced osteoblast cell viability and proliferation and promoted osteoblast differentiation by increasing ALP activity [38]. Reports have shown that higher vitamin C concentration promoted synthesis and growth of extracellular matrix proteins such as collagen-1 as well as non-collagenous protein like alkaline phosphatase, osteonectin, and osteocalcin [39]. These proteins are produced at various stages of differentiation of osteoblasts, which allows the extracellular matrix formation of calcified tissue, especially bone [40]. These reports validate our result on the positive influence of vitamin C on osteoblast proliferation. Reviewing all results, this work presents a controlled release of vitamin C as a promising alternative or adjunct treatment for post-tumor resection defect repair in addition to its osteogenic capabilities, suggesting clinical significance in bone tumor resection.

4. Summary

Localized and controlled delivery of vitamin C has been achieved from a load-bearing plasma-sprayed HAp coated cpTi implant and the effect of sustained drug release on in vitro osteoblast cell proliferation and osteosarcoma cell suppression was demonstrated. In vitro release study shows controlled delivery of vitamin C has been achieved from HAp coated cpTi implant for 21 days, while the data indicates that the release kinetics has been fitted well with the Korsmeyer-Peppas model. The effect of pH has been investigated to understand the dissolution behavior of HAp coating, and subsequently monitor the vitamin C drug delivery. Acidic buffer media at pH 5.0 results in the faster dissolution of the coating, which leads to a higher release of vitamin C ensuring localized burst release in acidic post-surgical or tumor microenvironment. On the other hand, physiological pH 7.4 causes much less degradation on the HAp coating surface and therefore exhibits slower but controlled delivery of vitamin C for a prolonged period. The presence of high dosage of vitamin C of 25 mM results in a significant inhibition in cell viability at as early as 3 days, and within 7 days vitamin C loaded implant exhibits 2.5 folds lesser MG-63 cell viability compared to control. However, the controlled release of vitamin C shows firm filopodial attachment with abundant osteoblast cell coverage on the implant surface at day 3 and day 7, which suggests its non-cytotoxicity towards healthy osteoblast cells. Although in vivo studies are necessary in future to better understand the clinical scenario of vitamin C on osteosarcoma, this present study suggests a promising drug delivery device, which can be utilized as a load-bearing bone graft after bone tumor surgeries to eradicate remaining cancer cells and promote faster bone growth.

Highlights.

  • Vitamin C was incorporated in a plasma sprayed HA coated load bearing implant.

  • Controlled release of vitamin C was achieved from HA coated implant for 21 days.

  • Higher release was observed in acidic pH due to higher degradation of HA coating.

  • Controlled release of 25mM vitamin C decreased osteosarcoma cell viability.

  • Vitamin C did not show any cytotoxicity towards healthy osteoblast cells.

Acknowledgment

Researchers would like to acknowledge financial assistance from NIH [Grant # 1R01-AR-066361].

Footnotes

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Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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