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Published in final edited form as: Carbohydr Polym. 2020 Sep 12;251:117079. doi: 10.1016/j.carbpol.2020.117079

Tanfloc/Heparin Polyelectrolyte Multilayers Improve Osteogenic Differentiation of Adipose-Derived Stem Cells on Titania Nanotube Surfaces

Roberta M Sabino 1, Gabriela Mondini 2, Matt J Kipper 1,3,4,*, Alessandro F Martins 4,5,6, Ketul C Popat 1,3,7,*
PMCID: PMC7717535  NIHMSID: NIHMS1631015  PMID: 33142622

Abstract

In this study, a surface modification strategy using natural biopolymers on titanium is proposed to improve bone healing and promote rapid and successful osseointegration of orthopedic implants. Titania nanotubes were fabricated via an anodization process and the surfaces were further modified with polyelectrolyte multilayers (PEMs) based on Tanfloc (a cationic tannin derivative) and glycosaminoglycans (heparin and hyaluronic acid). Scanning electron microscopy (SEM), water contact angle measurements, and X-ray photoelectron spectroscopy were used to characterize the surfaces. Adipose-derived stem cells (ADSCs) were seeded on the surfaces, and the cell viability, adhesion, and proliferation were investigated. Osteogenesis was induced and osteogenic differentiation of human ADSCs on the surfaces was evaluated via mineralization and protein expression assays, immunofluorescent staining, and SEM. The Tanfloc/heparin PEMs on titania nanotubes improved the rate of osteogenic differentiation of ADSCs as well as the bone mineral deposition, and is therefore a promising approach for use in orthopedic implants.

Keywords: Tannins, Heparin, Layer-by-layer technique, Titanium surfaces, Orthopedic implants, Osseointegration

Graphical Abstract

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1. INTRODUCTION

Titanium and its alloys are the most common biomaterial used for orthopedic and dental implants (Kaur & Singh, 2019). Titanium combines excellent mechanical properties, such as high strength, low density and moderate Young’s modulus, with great biocompatibility and resistance to corrosion (Planell & Navarro, 2009). In addition, titanium can have its properties easily modified by forming alloys or altering its surface, which makes it suitable for a wide range of biomedical applications (F. Zhang, Zhang, Zhu, Kang, & Neoh, 2008). However, the rate of failure for orthopedic and dental implants can be as high as 10% (Rastegari & Salahinejad, 2019; Smith, Dieppe, Vernon, Porter, & Blom, 2012). One of the major causes for device failure is the aseptic loosening of titanium implants, mostly due to poor osseointegration of the prosthesis into the bone (Dias-Netipanyj et al., 2019). Implants for knees, hips, and teeth need to withstand dynamic stresses, which create micromotion of the implant and can lead to the device loosening. Therefore, it is essential to develop novel biomaterial surfaces that promote better osseointegration, thus reducing implant failure and revision surgeries.

Osseointegration is the direct anchorage of a prosthesis to bone by the formation of bone tissue around the implant (T. Albrektsson, 2001). Rapid and stable osseointegration reduces risks of implant failure, and involves multiple biological processes, including preosteoblast adhesion, differentiation, and mineralization (Tang et al., 2018). To achieve osseointegration, an implant surface must be osteoinductive (i.e. promote osteoblast differentiation) and osteoconductive (i.e. promote new bone matrix deposition on its surface) (Frassica, Jones, Diaz-Rodriguez, Hahn, & Grunlan, 2019). Therefore, osteoconduction relies on an earlier successful osteoinduction, which ultimately leads to the implant osseointegration. The osteoinductivity of surfaces can be studied in vitro by investigating the osteogenic differentiation of stem cells in contact with the biomaterial surface (García-Gareta, Coathup, & Blunn, 2015). Adipose-derived stem cells (ADSCs) exhibit pluripotency in vitro, and are capable of differentiation into a variety of cell lineages, including osteoblasts. Since ADSCs demonstrate a substantial capacity for bone formation, they are an excellent model cell type to study osteogenic differentiation and osteoinduction on biomaterials (Cowden, Dias-Netipanyj, & Popat, 2019a).

Surface modifications have been proposed to enhance osseointegration on titanium implants. Altering the topography of the biomaterial surface to introduce micro and nanoscale features can improve the cellular responses (Cowden et al., 2019a; Manivasagam & Popat, 2020; Vishnu et al., 2019). Since bone cells in native tissue interact with nanoscale extracellular matrix elements, such as protein and minerals, surface nanotopography increases the adsorption of proteins and stimulates osteogenic differentiation (Souza et al., 2019). These nanoscale interactions predispose cells to adhere, proliferate, and differentiate on nanostructured surfaces (Pedrosa et al., 2019). Nanofabrication techniques recently investigated on titanium include formation of nanopores, nanofibers and nanotubes (Bai et al., 2018; Hyzy et al., 2016). Titania nanotube (NT) surfaces have emerged as a promising approach as they enhance biocompatibility, promote adhesion and differentiation of stem cells, improve antibacterial properties, and reduce immune responses on titanium (Cheng et al., 2018; Popat, Leoni, Grimes, & Desai, 2007).

The layer-by-layer (LbL) deposition of polyelectrolyte multilayers (PEMs) is another promising technique to improve the biological response to titanium-based surfaces is (Valverde et al., 2019). The LbL approach creates PEMs by alternating the adsorption of polycationic and polyanionic layers onto solid surfaces (Barrantes, Wengenroth, Arnebrant, & Haugen, 2017). Tanfloc (TA) is a cationic polyphenol that our research group has recently investigated as a biomaterial (da Cruz et al., 2020; Lopes et al., 2019). TA is an amino-functionalized tannin derivative prepared from condensed tannins using a Mannich’s type reaction with formic acid and ammonium chloride (D. P. Facchi et al., 2017). TA can increase the biocompatibility, antithrombogenic and antimicrobial properties of biomaterials (da Câmara et al., 2019; Facchi et al., 2020; Martins et al., 2018). TA-based PEMs improve the hemocompatibility, cell adhesion, and antibacterial activity of solid substrates (Sabino et al., 2020; da Câmara et al., 2020). The osteoinductive properties of TA-based surfaces has never been investigated. In this work, TA-based PEMs on NT were fabricated by first changing the surface topography of titanium to make titania nanotubes. Then, the NT surface was modified via the LbL technique using the polyelectrolytes TA (polycation), and heparin (HP, polyanion) or hyaluronic acid (HA, polyanion) to create PEMs with 5-layers on NT. We have previously used polysaccharide-containing PEMs to modify the surfaces of allograft bone, showing that these are suitable for promoting stem cell adhesion, reducing bacterial attachment, and improving inflammatory responses in vivo (Almodóvar et al., 2010; Kipper et al., 2014; Romero et al., 2015). Polysaccharide based PEMs can also be used to control growth factor delivery to guide cell behavior (Almodóvar et al., 2010; Boddohi & Kipper, 2010; Lin et al., 2018; Place, Kelly, & Kipper, 2014; Place, Sekyi, & Kipper, 2014; Volpato et al., 2012). Based on this work, we hypothesize that the combination of polysaccharide-containing Tanfloc PEMs can improve osteoinduction of ADSCs on titania nanotube surfaces.

Surfaces were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), contact angle goniometry, and stability tests. ADSCs were cultured on the surfaces, and their viability, adhesion and proliferation were evaluated after 4 and 7 days of cell culture. Osteogenesis was then induced and the osteogenic differentiation of ADSCs was assessed via mineralization and protein expression assays, immunofluorescent staining, and SEM. The TA/HP PEMs on NT significantly improve osteogenic differentiation of ADSCs and bone mineral deposition compared to unmodified NT, indicating potential for enhanced osseointegration.

2. MATERIALS AND METHODS

2.1. Fabrication of titania nanotubes (NT) modified with PEMs

NT surfaces were fabricated using an anodization technique on titanium sheets (0.4 mm thick) as described elsewhere (Sabino, Kauk, Movafaghi, Kota, & Popat, 2019). Tanfloc (TA, Tanac SA, approximately 60 kDa, characterized as described in the Supplementary Material) was purified by dialysis as previously reported (Martins et al., 2018). Solutions of TA, HP (Celsus Laboratories, 14.4 kDa), and HA (Sigma, 1.5–1.8 MDa) were made in acetic acid-acetate buffer (pH 5.0 and 0.2 M) at 1.0 mg/ml concentration. NT surface modification with 5-layer PEMs was previously described and is detailed in the Supplementary Material (Sabino et al., 2020). The PEMs deposited on NT surfaces are denoted NT(TA-HP) and NT(TA-HA) (Fig. 1).

Fig. 1.

Fig. 1.

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Schematic of PEM fabrication on titania NT and the polyelectrolyte structures (R = H or OH).

2.2. Surface characterization

The morphology of the surfaces was characterized using SEM. The surfaces were coated with 10 nm of gold and imaged at 15 kV. The ImageJ software was used to measure the nanotubes’ diameters. The static contact angles were measured with 7 μl of DI water on the surfaces using a Ramé-Hart Model 250 goniometer. The chemical compositions of the surfaces were characterized using XPS. Survey-scan spectra were obtained from 0 to 1100 eV at a pass energy of 187 eV, and high-resolution spectra of carbon C1s peak were obtained at a pass energy of 10 eV. Peak-fit analysis was performed using MultiPak and Origin software.

2.3. ADSC culture

Prior to biological assays, all the surfaces were sterilized by incubating with 70% ethanol for 5 min, followed by 3 rinses and incubation with sterile PBS for 30 min. Human ADSCs isolated from adipose tissue and at passage three were obtained from Dr. Kimberly Cox-York’s laboratory at Colorado State University. The protocol for ADSC isolation from healthy individuals was approved by Colorado State University Institutional Review Board. The cells were cultured at 37 °C and 5% CO2 in growth media composed of α-MEM Media (HyClone™) with 10% (v/v) Fetal Bovine Serum (FBS, Gibco) and 1% (v/v) penicillin/streptomycin (Corning) (Ramin et al., 2019). The cells were seeded on the sterilized NT surfaces at a final concentration of 2.0 × 104 cells/ml.

2.4. ADSCs viability, adhesion, and proliferation

ADSC viability was evaluated using CellTiter-Blue assay (Promega) after 4 and 7 days of cell culture. The assay was previously described and is detailed in the Supplementary Material (Ramin et al., 2019). The cell adhesion and proliferation on the surfaces were characterized using fluorescence microscopy. After 4 and 7 days of culture, the media was removed, and the surfaces were rinsed with PBS. The cells adhered on the surfaces were fixed in 3.7% formaldehyde (Fisher, 3.7%) in PBS for 15 mins, followed by three rinses (5 min each) with PBS. Adhered ADSCs were permeabilized by incubation with 1% Triton X-100 (Fisher, 98%) in PBS for 3 min and rinsed twice with PBS (de Oliveira et al., 2020). The surfaces were then incubated in 70 nM of rhodamine phalloidin (Cytoskeleton, Inc.) in PBS for 20 min in a dark environment. The nuclear stain DAPI (300 nM, ThermoFisher Scientific) was added, and after 5 min the surfaces were rinsed with PBS twice and then imaged using a fluorescence microscope (Zeiss). To obtain the quantity of ADSCs adhered on the surfaces, the number of stained nuclei (DAPI) was counted using ImageJ software. Cell morphology on the surfaces was also investigated by SEM. The fixation process was previously described and is detailed in the Supplementary Material (de Souza et al., 2020).

2.5. Osteogenic differentiation of ADSCs

After 7 days of ADSC culture on the surfaces, osteogenesis was induced using osteogenic differentiation media, consisting of growth media supplemented with 10−8 M dexamethasone (Sigma, 98%), 50 μg/mL of ascorbic acid (Sigma, 100%), and 6 mM β-glycerol phosphate (Sigma, 98%). The differentiation media was changed every other day for 3 weeks. After 1 and 3 weeks of induced osteogenic differentiation, the following quantitative assays were performed to determine the osteoinductivity of the surfaces: total protein content, alkaline phosphatase (ALP) activity, and calcium concentration. The assays were previously described and are detailed in the Supplementary Material (Cowden, Dias-Netipanyj, & Popat, 2019b).

The osteogenic differentiation of ADSCs on the surfaces was also analyzed using immunofluorescent staining of the cells for the bone protein osteocalcin. After 1 and 3 weeks of induced osteogenesis, the media was removed, and the surfaces rinsed twice with PBS. The adhered cells were fixed and permeabilized as described in Section 2.4. The surfaces were then incubated in 10% bovine serum albumin (BSA, Sigma) for 30 min, followed by incubation in osteocalcin primary antibody (Santa Cruz Biotechnology) at a dilution of 1:100 in 1% BSA for 60 min. After two rinses with PBS, the surfaces were incubated with secondary antibody-FITC (Santa Cruz Biotechnology) at a dilution of 1:200 in 1% BSA for 45 min, followed by two rinses with PBS. Then, the surfaces were incubated in rhodamine phalloidin and DAPI nuclear stain, as described in Section 2.4. The surfaces were imaged by fluorescence microscopy. The presence of osteocalcin on each surface was obtained by measuring the percentage coverage area of stained osteocalcin using ImageJ software. The osteocalcin percentage area was normalized to the total number of nuclei on each surface.

The cell morphology and mineral deposition on the surfaces were also characterized using SEM. After 1 and 3 weeks of induced osteogenesis, ADSC were fixed and dehydrated using the same process described in Section 2.4. Before the SEM imaging, the surfaces were coated with gold and imaged as described in Section 2.2.

2.6. Statistical analysis

SEM images and XPS analysis were performed on at least 2 different samples of each surface type. Contact angle and stability tests were carried out using 2 droplets per sample on 3 different samples of each surface type (nmin = 6). All cell studies were carried out with nmin = 3 for qualitative analyses and nmin = 5 for quantitative analyses. The quantitative results were analyzed via analysis of variance (ANOVA) and Tukey tests using the GraphPad Prism software (p < 0.05).

3. RESULTS AND DISCUSSION

Titanium and its alloys have been mainly used as biomaterials for orthopedic and dental implants due to their excellent mechanical properties, biocompatibility, and resistance against corrosion (Kaur & Singh, 2019). However, implant failure is still a huge problem due to biological issues, including poor osseointegration, microbial infection, and inflammation (Cowden et al., 2019b). Therefore, the osseointegration enhancement of a biomaterial plays a key role in bone repair and tissue regeneration (Zhou et al., 2018). To minimize the chances of implant failure, several approaches have been proposed to modify the surface chemistry and topography of titanium (Gregurec et al., 2016; K. Zhang et al., 2016; X. Zhang et al., 2018). Our recent work showed that the NT modification with TA-based PEMs significantly enhances the antithrombogenic and antibacterial properties of NT surfaces (Sabino et al., 2020). This work showed that TA/HP and TA/HA PEMs (5-layers) on NT significantly reduced the adhesion and proliferation of bacteria (P. aeruginosa and S. aureus) after 24 h of incubation, and decreased fibrinogen adsorption, Factor XII activation, and platelet adhesion and activation in comparison with unmodified NT surface (Sabino et al., 2020). These properties are essential to prevent blood clotting and biofilm formation on implanted devices. However, the osteoinductivity of TA-based PEMs has never been investigated. In this study, we expand the characterization of the NT surfaces modified with PEMs. The surfaces are characterized by SEM, contact angle goniometry, and XPS; their stability in physiological conditions is also evaluated. Cell viability, attachment, proliferation, and differentiation of ADSCs into bone cells was also evaluated.

3.1. Surface characterization

SEM was used to characterize the surface morphology of NT and NT modified with PEMs (Fig. 2). All surfaces have uniform and vertically oriented nanotubes. The SEM images show some changes in morphology for NT(TA- HA) in comparison with the others, although the nanotubes are still visible. No changes in NT average diameter (112 ± 11 nm) were observed before and after surface modification. This agrees with our previous study, showing that 5-layer PEMs do not achieve complete coverage of the NT surfaces (Sabino et al., 2020). We choose to use 5-layer PEMs to achieve surface modification, while maintaining the nanoscale topography of the surface. These nanofeatures may increase their surface biocompatibility by mimicking nanoscale behavior of cell-extracellular matrix interactions (de Almeida et al., 2020).

Fig. 2.

Fig. 2.

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Representative SEM images of NT surfaces before and after modification with PEMs. The images were taken at 10,000× magnification.

Contact angle measurements were used to characterize the wettability of all surfaces. All of the surfaces here are hydrophilic, defined as having a water contact angle θ < 90° (Wang et al., 2016) (Fig. 3). NT surfaces are considered superhydrophilic (θ < 10°) as the water penetrates by capillary forces in the tubular structures (Lai et al., 2008). NT(TA- HP) shows higher hydrophilicity than NT(TA- HA) because HP possesses a high number of sulfate groups on its structure (Follmann et al., 2012).

Fig. 3.

Fig. 3.

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Static water contact angles for different NT surfaces. No significant differences in contact angle between NT and NT(TA-HP) was observed. (*) represents p < 0.05 and (**) p < 0.01.

XPS was used to evaluate the chemistry of different NT surfaces. Survey spectra of all surfaces have titanium (Ti2p), oxygen (O1s), and carbon (C1s) peaks (Fig. 4a). The small C1s on NT surfaces arises due to adventitious carbon (impurities). The C1s peak intensity is greater on and the Ti2p peak is substantially reduced in the spectra of NT(TA-HP) and NT(TA-HA), indicating nearly complete coverage of the NT surface by the PEMs. Both PEM-modified surfaces also have nitrogen (N1s). The N1s signal arises from the amino groups of the TA and from the glucosamine residues in the glycosaminoglycans (da Cruz et al., 2020). NT(TA- HP) has sulfur (S2p and S2s) peaks, due to the sulfate groups in HP, which confirms the addition of heparin. High-resolution C1s spectra are shown in Fig. 4b. As expected, NT(TA- HP) and NT(TA- HA) have increased C1s peaks, including C–O and C–N groups from the polyelectrolytes’ structures (Table 1 in Supplementary Material). The C1s spectra also confirm that NT are successfully modified with PEMs, as O–C=O, N–C=O, C–O, C–N, and C–C bonds are characteristic of the polyelectrolytes’ chemical structures. Both survey and high-resolution spectra confirm the successful medication of NT surfaces. Before the PEM deposition, the NT surfaces were treated with oxygen plasma to form hydroxyl groups, allowing the with polycation, TA to bind. The PEMs were then constructed by alternating polycation-polyanion deposition onto the NT surface by electrostatic self-assembly.

Fig. 4.

Fig. 4.

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(a) XPS survey scans and (b) High resolution C1s scans for different surfaces.

Prior to the biological assays, the stability of different NT surfaces was evaluated over a 28-day period using the method described elsewhere (Sabino et al., 2019). The results are detailed in the Supplementary Material and show that the PEMs are stable under buffer conditions for 28 days.

3.2. ADSCs viability, adhesion, and proliferation

ADSCs have attracted great attention for orthopedic biomaterial studies. ADSCs are easily obtained from human fat tissue, with a high yield of cells and minimal donor-site morbidity (Bombaldi de Souza et al., 2020). They can differentiate into several cell lineages, including osteoblasts, making them a good alternative to bone-marrow derived mesenchymal stem cells (BMSCs). BMSCs are commonly used for investigation of biomaterial-cell interaction and osteoinduction (Halim et al., 2019); however, their harvest is invasive and produces a low BMSC yield (Cowden et al., 2019a). Since ADSCs show bone formation capacity in vitro, they were used in this study. Cell viability was quantified after 4 and 7 days using the CellTiter-Blue assay. The viable cells reduce the resazurin to resorufin by dehydrogenase enzymes (Madruga et al., 2020). Thus, a higher percentage of CellTiter-Blue reduction indicates greater cell viability. The cell viability results were normalized using the positive control (tissue-culture polystyrene). No significant difference in cell viability are observed after 4 days of culture (Fig. 5a), indicating similar initial cell attachment on all surfaces. After 7 days, NT(TA- HA) promoted a significant decrease in the cell viability compared to NT. No significant difference was observed between NT and NT(TA- HP) after 7 days of culture, indicating that these surfaces presented higher metabolic activity of ADSCs than NT(TA- HA).

Fig. 5.

Fig. 5.

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(a) CellTiter-Blue ADSC viability assay after 4 and 7 days of culture. Results are normalized using the positive control on tissue-culture polystyrene. (b) Representative fluorescence microscopy images of ADSCs stained with DAPI (blue) and rhodamine phalloidin (red) after 4 and 7 days of proliferation. (c) Cell count per area after 4 and 7 days of ADSC culture. No significant difference is observed on day 4 between all NT surfaces. Cell counts on NT and NT(TA-HP) are significantly higher at day 7 than at day 4. No significant difference in cell counts was observed on NT(TA-HA) between days 4 and 7. * Represents p < 0.05; *** represents p < 0.001; **** represents p < 0.0001.

Initial adhesion and proliferation of stem cells on biomaterial surfaces are crucial as they influence osteogenic differentiation, and consequently the implant long-term stability (Bombaldi de Souza et al., 2020). The ability of scaffolds to support adhesion, proliferation, and spreading of ADSCs was investigated using SEM and fluorescence microscopy images (Fig. 5b, and Fig 6). After 4 days of cell culture, no significant difference in the cell adhesion imparted by the surfaces is observed (Fig. 5c). However, the NT surface has a significantly higher number of ADSCs after 7 days compared to both PEM-modified surfaces. This indicates that unmodified NT surfaces impart higher stem cell proliferation than NT modified with TA-based PEMs. It has been shown that TA-based PEMs have lower protein adsorption on the surface (da Câmara et al., 2020; Sabino et al., 2020). Thus, these surfaces will result in lower ADSCs adhesion and will affect subsequent cell proliferation (Arima & Iwata, 2007). SEM images show that ADSCs spread similarly on all NT surfaces (Fig. 6). Therefore, although the number of adhered cells is larger on unmodified NT after 7 days, both NT(TA- HP) and NT(TA- HA) surfaces enable similar sustainable cell growth and proliferation as unmodified NT.

Fig. 6.

Fig. 6.

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Representative SEM images of cells on NT surfaces after 4 and 7 days of ADSCs culture. The images were taken at 1,000× magnification. For a better visualization, the cells on the NT surfaces are post blue-colored.

3.3. Osteogenic differentiation of ADSCs

After 7 days of ADSC culture, the differentiation of cells into osteoblasts was induced by supplementing the growth media with dexamethasone, ascorbic acid, and β-glycerol phosphate (Cowden et al., 2019b). Dexamethasone enhances RUNX2 activity (a transcription factor related to osteoblast differentiation); ascorbic acid increase the secretion of collagen type I; and β-glycerol phosphate is the phosphate source for hydroxyapatite production (Langenbach & Handschel, 2013). Osteogenesis is crucial, as it predicts the capacity for tissue regeneration and long-term stability of orthopedic implants, playing a vital role in preventing device failure. After 1 and 3 weeks, cell functions associated with osteoblast differentiation (ALP activity, calcium deposition, and osteocalcin deposition) were evaluated. Mineralization was also characterized from SEM images.

ALP is an enzyme in bone matrix vesicles, and is considered indispensable to mineralization (Sugawara, Suzuki, Koshikawa, Ando, & Iida, 2002). Its activity is related to calcification, as the ALP level is high when the level of inorganic phosphate, a component of the bone mineral phase, increases (de Souza et al., 2020). Therefore, ALP activity has a peak just before mineralization begins and is considered as an early indicator of osteoblast differentiation (Cowden et al., 2019b). No significant difference in normalized ALP activity is found after 1 week of induced differentiation (Fig. 7). After 3 weeks, NT(TA- HP) and NT(TA- HA) have significantly higher ALP activity than the NT surface. The ALP production confirms that the PEM-modified NT surfaces are more supportive of osteoblast differentiation than the unmodified NT surfaces. Although the initial adhesion and proliferation of cells (Figure 5) is important, the key factor for an enhanced osseointegration is how fast and effectively the stem cells differentiate into osteoblasts.

Fig. 7.

Fig. 7

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ALP activity normalized by total protein content (micro BCA assay). ALP and BCA assays were performed after 1 and 3 weeks of induced osteogenesis. (*) represents p < 0.05.

Calcium content on the surfaces was quantified, as it is one of the main components of hydroxyapatite, produced during the mineralization process (Zhao, Zhou, Li, Zou, & Du, 2018). Mineralization is the process by which bone cells produce hydroxyapatite, the principal inorganic component of the bone (Anderson, 2003). Calcium deposition occurs late in the osteogenesis process, when osteoblasts produce enzymes that cleave proteoglycans to release calcium and phosphate ions to form hydroxyapatite (Cowden et al., 2019b) (de Souza et al., 2020). Therefore, calcium deposition is an indicator of the osteoconductive capacity of a biomaterial, and the later stage of differentiation of ADSCs. No significant difference is observed between groups after 1 week, with a significantly higher calcium content in all samples at week 3, compared to week 1 (Fig. 8). NT(TA- HP) promotes significantly higher calcium deposition compared to unmodified NT surfaces after 3 weeks of induced osteogenesis (Fig. 8). This is consistent with ALP activity, as an increased marker for early stage differentiation on TA- based PEMs promotes higher calcium accumulation on these surfaces at a later time.

Fig. 8.

Fig. 8

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Calcium deposited on the surfaces normalized by total protein content. Calcium and total protein assays were performed after 1 and 3 weeks of induced osteogenesis. (**) represents p < 0.01.

To form the organic matrix for mineralization, osteoblasts will produce several collagenous and non-collagenous proteins, including osteocalcin (Florencio-Silva, Sasso, Sasso-Cerri, & Simões, 2015). Osteocalcin is a late marker of differentiation produced exclusively by osteoblasts and is involved in bone matrix formation (Cowden et al., 2019b). Immunofluorescence images show that osteocalcin significantly increases on all surfaces after 3 weeks of induced differentiation when compared to week 1 (Fig. 9). The percentage area coverage of osteocalcin is significantly higher for NT(TA- HP) compared to the other surfaces, with an increase of approximately 80% (Fig. 9b). This agrees with ALP activity and calcium concentration results, showing that NT(TA- HP) improve osteoblast differentiation when compared to the other surfaces.

Fig. 9.

Fig. 9.

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(a) Representative immunofluorescence microscopy images of ADSC after 1 and 3 weeks of induced osteogenesis. Green stain represents osteocalcin. (b) Percentage area coverage of osteocalcin normalized by number of nuclei after 1 and 3 weeks of induced osteogenesis. The results on week 3 are significantly higher from week 1 across all treatments. (****) represents p < 0.0001.

The mineral deposition and morphology of ADSCs on surfaces were analyzed using SEM after 1 and 3 weeks of induced osteogenesis. All surfaces are covered by ADSCs and induce mineral deposition after 3 weeks. However, NT(TA- HP) have more mineral deposits (Fig. 10). These deposits may be hydroxyapatite, a mineral mainly formed by calcium and phosphorus, and matrix vesicles (Cowden et al., 2019b). Matrix vesicles are membrane-invested particles where the first crystals of bone mineral are formed (Anderson, 2003). SEM results agree with ALP, calcium, and osteocalcin outcomes, confirming that NT(TA- HP) supports higher mineral deposition, also indicating enhanced osteogenic differentiation on this surface.

Fig. 10.

Fig. 10.

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Representative SEM images of mineral deposition and ADSCs after 1 and 3 weeks of induced osteogenesis. The images were taken at 1,000× magnification.

Unmodified NT surfaces support the highest number of adhered ADSCs. However, NT(TA- HP) promotes stronger osteogenic differentiation than unmodified NT. Tannic acids can enhance the osteoinduction through binding calcium ions, one of the most essential metal ions involved in osteogenesis (Li et al., 2020; Xu, Neoh, & Kang, 2018). TA is a cationic derivative of condensed tannins, comprising a high content of phenolic groups, which could bind metal ions, such as calcium. TA also contains amine groups (pKa = 6.0), which can also bind calcium ions (Valcarce et al., 1993). These chemical traits may improve osteogenic differentiation of human ADSCs. Although both TA- based PEMs show enhanced osteogenesis when compared to unmodified NT, TA/HP PEMs on NT impart the best results for osteoblast differentiation. This could be because many proteins associated with osteoblast differentiation contain heparin-binding domains (Benoit, Durney, & Anseth, 2007). Therefore, when interacting with numerous proteins related to osteogenesis, such as vitronectin and bone morphogenetic proteins (BMPs), heparin can selectively activate desired cell functions and improve osteogenic differentiation (Benoit & Anseth, 2005; Kim, Kim, Jung, Hong, & Lee, 2014). The association between TA and HP provides a surface with suitable properties to enhance osteogenic differentiation on a titanium-based biomaterial.

4. CONCLUSIONS

In this work, TA/polysaccharide PEMs were prepared on titania NT to improve cell behaviors associated with osseointegration on titanium implants. Osseointegration is critical to prevent aseptic loosening, thus promoting the success of orthopedic and dental implants. The NT surfaces were successfully modified with PEMs and shown to be stable under phosphate buffer exposure over 28 days. NT surfaces modified with the TA/HP PEMs induce higher alkaline phosphatase activity, mineral deposition, osteocalcin and calcium concentration compared with unmodified NT, indicating enhanced osteoinductivity toward human ADSCs. The polyphenol and amine moieties in TA may promote bone healing. Also, HP plays an important role due to binding with signaling proteins involved in osteogenesis. For the first time, we show that the titania NT can be modified with TA and HP to promote stem cell differentiation. These surfaces may improve early-stage osseointegration of implants, thus reducing the risk of device failure due to aseptic loosening.

Supplementary Material

mmc1

ACKNOWLEDGEMENTS

The authors thank Dr. Kimberly Cox-York for isolating and donating the human adipose derived stem cells. Research reported in this publication was supported by National Heart, Lung and Blood Institute of the National Institutes of Health under award number R01HL135505 and R21HL139208.

Footnotes

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