3D printed β-tricalcium phosphate versus synthetic bone mineral scaffolds: A comparative in vitro study of biocompatibility

Abstract

BACKGROUND:

β-tricalcium phosphate (β-TCP) has been successfully utilized as a 3D printed ceramic scaffold in the repair of non-healing bone defects; however, it requires the addition of growth factors to augment its regenerative capacity. Synthetic bone mineral (SBM) is a novel and extrudable carbonate hydroxyapatite with ionic substitutions known to facilitate bone healing. However, its efficacy as a 3D printed scaffold for hard tissue defect repair has not been explored.

OBJECTIVE:

To evaluate the biocompatibility and cell viability of human osteoprecursor (hOP) cells seeded on 3D printed SBM scaffolds via in vitro analysis.

METHODS:

SBM and β-TCP scaffolds were fabricated via 3D printing and sintered at various temperatures. Scaffolds were then subject to qualitative cytotoxicity testing and cell proliferation experiments utilizing (hOP) cells.

RESULTS:

SBM scaffolds sintered at lower temperatures (600 °C and 700 °C) induced greater levels of acute cellular stress. At higher sintering temperatures (1100 °C), SBM scaffolds showed inferior cellular viability relative to β-TCP scaffolds sintered to the same temperature (1100 °C). However, qualitative analysis suggested that β-TCP presented no evidence of morphological change, while SBM 1100 °C showed few instances of acute cellular stress.

CONCLUSION:

Results demonstrate SBM may be a promising alternative to β-TCP for potential applications in bone tissue engineering.

Keywords

3D printing direct inkjet writing bone regeneration bioceramics synthetic bone mineral

1. Introduction

Congenital disorders, infections, tumors, or traumas often warrant surgical intervention with large bone defects [1–4]. Irrespective of the cause of the bone defect, the aim of surgery is to restore native anatomic morphology as well as function. In defects that are critically-sized (CSD), or unable to heal spontaneously, repair cannot be achieved without the use of grafts [5]. Synthetic materials, more specifically bioceramics, such as calcium phosphates (CaP), calcium carbonates, calcium sulfates, and bioactive glasses, have garnered attention as alternatives to autografts, allografts and xenografts. Furthermore, they exhibit excellent biocompatibility, high elastic modulus, as well as favorable osteointegrative and osteoconductive properties [6–8]. Despite these advantages, commercially available bioceramic alloplasts are commonly in particulate or block form and are thus limited by their non-customizable shape, size, composition, and lack of microstructure known to facilitate bone regeneration [7,9,10]. For these reasons, research focus has shifted to fabrication of 3D-printed (3DP) bioceramic scaffolds for potential applications in craniomaxillofacial and orthopedic surgery [11,12].

Additive manufacturing via 3D-printing has led to significant advancements within the field of bone tissue engineering (BTE) as it allows for customization of scaffold architecture to precisely fit-and-fill patient-specific defects [13]. Furthermore, alterations to bioceramic scaffold’s surface topography, internal porosity, and scaffold loading with regenerative pharmaceuticals or exogenous growth factors have been shown to augment bone formation [10,14,15]. Direct Inkjet Writing (DIW) is an extrusion-based 3D printing technique that is compatible with bioceramic colloidal gels [16,17]. Scaffold fabrication occurs via heatless, pressurized syringe pumps that extrude desired build and support materials in a layered fashion according to a 3D model generated by Computer Aided Design (CAD) [16,17]. During post-processing, support material is removed and the final bioceramic scaffold is sintered to improve mechanical strength by densification [18]. Relative to other bioceramics, DIW of β-tricalcium phosphate (β-TCP) has been thoroughly explored in literature as it possesses more favorable properties for bone regeneration [19–21].

β-TCP is similar in composition to the mineral phase of bone, and is associated with a well-known safety profile and biocompatibility [12]. Additionally, preclinical studies have shown scaffold degradation occurs between 6- to 18- months which is superior to that of hydroxyapatite (∼1% per year) for hard tissue repair [22,23]. Many in vivo studies have found successful healing of critical-sized calvarial, alveolar, mandibular, and radial defects of skeletally immature or mature rabbit, ovine, and porcine models using β-TCP fabricated scaffolds [12,14,23–31]. Additionally, mesenchymal stem cells (MSCs), endothelial progenitor cells, platelet concentrates, recombinant human bone morphogenetic protein (rh-BMP2), and dipyridamole have all been investigated as potential osteogenic additives [12,14,23,25–33]. However, the addition of such biologic components to scaffolds poses challenges with the U.S. Food and Drug Administration (FDA) product approval process prior to clinical use [34]. An alternative strategy to enhance osteogenic capacity has been detailed to be through ionic substitution with bioactive ions which chemically constitute human bone [35,36]. Successful ion substitution, by way of avoiding categorization as a “combination product”, would expedite the FDA approval process for bench-to-bedside translation of such biomimetic scaffolds. Of note, LeGeros et al. synthesized an ion substituted CaP biomaterial, coined synthetic bone mineral (SBM), for investigation of its potential in bone healing [37].

To elaborate, SBM is a novel carbonate hydroxyapatite with magnesium (Mg²⁺) and zinc (Zn²⁺) ion substitutions. Individually, these ions have been identified as key components of bone mineralization and repair [35]. Furthermore, SBM possesses a similar carbonate content to biological bone apatite. Previous in vivo studies have demonstrated successful treatment outcomes when administered as an oral supplement or injected subcutaneously, SBM increased bone mass in animals fed mineral deficient diets or those who were ovariectomized [38–42]. More recently, a study performed in 2021 not only confirmed its printability with DIW, but also found that SBM scaffolds exhibited similar mechanical properties to those fabricated with β-TCP after sintering [35]. It is important to note that the benefit of increased mechanical strength associated with higher sintering temperatures is not without compromise to SBM’s unique carbonate content. This change in carbonate content and its effect on cellular viability of SBM remains unknown. In addition, to our knowledge, β-TCP and SBM have not been compared in an in vitro study for future consideration of SBM in critical-sized bone defect repair. This study hence aimed to evaluate biocompatibility of 3D printed (DIW-based) SBM scaffolds and to compare in vitro performance to analogous β-TCP scaffolds. The postulated null hypothesis of the present study was that there would be no difference in the viability of cells seeded on 3D printed SBM and β-TCP scaffolds.

2. Materials and methods

2.1. Scaffold design and manufacturing

Cuboidal scaffolds were designed (Fig. 1A) using custom CAD software (RoboCAD v6, 3D Ink LLC, Tulsa, OK, USA). For scaffold fabrication (Fig. 1B), printing was performed using a custom-built DIW printer (3D Inks, Tulsa, OK, USA). The colloidal gels of SBM and β-TCP were made in accordance with well-established formulations described in previous work [20,38]. In brief, SBM or β-TCP powders were mixed with precalculated amounts of a dispersant (ammonium polyacrylate), viscosifier (hydroxypropyl methyl cellulose) and anti-flocculant (polyethyleneimine) in a planetary mixer to ensure adequate mixing. B-TCP or SBM colloidal gels were loaded into 3 cc leur-lock syringe equipped with a general-purpose dispensing tip with an outlet diameter 250 μm (Nordson, East Providence, RI, USA). Scaffolds were printed within a paraffin oil bath to ensure uniform drying of printed constructs. During post-processing, β-TCP scaffolds (n = 60) were sintered to 1100 °C (β-TCP 1100 °C) and SBM Scaffolds were sintered to (i) 600 °C (SBM 600 °C), (ii) 700 °C (SBM 700 °C), or (iii) 1100 °C (SBM 1100 °C), (n = 60/group), respectively as indicated in the literature [35].

Fig. 1.

(A) Cuboidal scaffold designed using RoboCAD v6 software. (B) Final SBM cuboidal scaffold after sintering at 1100 °C.

2.2. Human osteoprecursor cell preparation

Human osteoprecursor (hOP) cells were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Embryonic Stem-cell Fetal Bovine Serum, and 1% Antibiotic/Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). Cells were stored at 37 °C under 5% CO₂ in a humidified incubator. After 48 h, cells were washed with Phosphate Buffered Saline (ThermoFisher Scientific, Waltham, MA, USA), and fresh media was supplemented ever 48 h. When ∼70% confluence was achieved, cells were detached from the culture plate using Trypsin (ThermoFisher Scientific, Waltham, MA, USA), and subsequently counted using an automated cell counter (Countess II FL, Life Technologies, Waltham, MA, USA).

2.3. Biocompatibility testing

A direct contact protocol was followed for qualitative cytotoxicity testing [43]. Prior to cell seeding, scaffolds were sterilized in an autoclave. Each well of a 48-well plate received 10⁵ osteoprogenitor cells re-suspended in 1 mL of culture media, followed by direct scaffold placement. Cells surrounding the scaffold were visualized for signs of morphological change using light microscopy (Leica Dmil LED and Leica Application Suite X software, Leica Biosystems, Wetzlar, Germany). After 48 h, scaffolds (SBM 600 °C, SBM 700 °C, SBM 1100 °C and β-TCP 1100 °C) seeded with hOP cells were transferred to a 48-well plate in preparation for the cell proliferation experiment.

2.4. Cell proliferation experiment

Cell proliferation was assessed utilizing a PrestoBlue^TM Cell Viability Fluorometric Assay (Life Technologies, Carlsbad, CA, USA) performed according to manufacturer’s instruction at 4 time-points (48 h, 72 h, 120 h, and 168 h). The fluorometric result was obtained after 30 m of incubation (37 °C) using a microplate reader (FilterMax 5, Molecular Devices, San Jose, CA, USA). This resazurin-based solution reduced to resorufin within living cells, a highly fluorescent compound with a maximum absorbance of 570 nm. Thus, the number of relative fluorescent units was directly proportional to the number of viable cells in culture. A monolayer of hOP cells seeded on a 48-well plate immersed in cell culture media without a scaffold served as the negative control.

2.5. Statistical analysis

Statistical evaluation of cell proliferation was performed using one-way analysis of variance (ANOVA) and Scheffé’s post hoc analyses for the four different groups (β-TCP 1100 °C, SBM 600 °C, SBM 700 °C, and SBM 1100 °C) at each time-point (48 h, 72 h, 120 h, and 168 h). The results are presented as mean values with corresponding 95% confidence intervals (mean ± CI). Statistical analyses of data were performed using SPSS version 29 (IBM Corp., Armonk, NY, USA), with p < 0.05 indicating significance.

Fig. 2.

Microscopic images of hOPs captured after direct contact of 3D printed scaffolds: SBM 600 °C (A), 700 °C (B), 1100 °C (C) and β-TCP 1100 °C (D) following 2 h of incubation. Higher magnification images of hOPs captured after direct contact of 3D printed scaffolds: SBM 600 °C (A.1), 700 °C (B.1), 1100 °C (C.1) and β-TCP 1100 °C (D.1).

3. Results

3.1. Biocompatibility testing

Figure 2 includes representative microscopic images of hOP cells in direct contact with SBM 600 °C, SBM 700 °C, SBM 1100 °C and β-TCP 1100 °C after a 2-h exposure period. Cells within the SBM 600 °C and 700 °C groups, exhibited an expanded, spherical morphology indicative of acute cellular swelling. This is better visualized in Fig. 3A.1 (red arrows). In contrast, a mix of morphologically normal and acutely swollen cells were observed in those subjected to SBM 1100 °C, (Fig. 3B.1 – yellow and red arrows, respectively), while cells exposed to β-TCP 1100 °C exhibited no signs of stress, appearing normal with their native morphology.

Fig. 3.

Microscopic images of SBM scaffolds in direct contact with hOPs after 2 h of exposure. (A) SBM 700 °C surrounded by acutely swollen cells (A.1, red arrows). (B) SBM 1100 °C surrounded by acutely swollen (B.1, red arrows) and other morphologically normal (B.1, yellow arrows) cells.

3.2. Cell proliferation experiment

Cell proliferation was measured after 48-, 72-, 120-, and 168- h (Fig. 4A–D), respectively. hOP cells cultured over SBM scaffolds sintered at all temperatures presented significantly lower rates of proliferation relative to the negative control (p < 0.001) and β-TCP scaffolds (p < 0.002) across all time-points. No significant differences were observed between SBM groups at the earlier time-points (48-, 72- and 120- hours) (p > 0.483). After 168 h of incubation, SBM 1100 °C exhibited greater cell viability than SBM 600 °C (p < 0.001), however relative to SBM 700 °C, no significant differences were observed (p = 0.064) (Fig. 4D). Of note, cell viability of β-TCP 1100 °C was significantly higher compared to both SBM 1100 °C and negative control after 168 h (p < 0.05).

Fig. 4.

Cell viability (%) of hOP cells seeded over SBM 600 °C, 700 °C, 1100 °C and β-TCP 1100 °C after (A) 48 h, (B) 72 h, (C) 120 h, and (D) 168 h of incubation. The control is depicted as a dashed line, representing 100% of hOP cell proliferation. * is p ≤ 0.05; ** is p ≤ 0.01; *** is p ≤ 0.001; **** is p ≤ 0.0001.

4. Discussion

Due to the increased popularity of 3D printing in the synthesis of scaffolds for defect repair, new methods of enhancing their bioactivity are being explored. For decades, the most common material utilized for alloplastic or synthetic grafts for bone tissue regeneration has been β-TCP. The superior biocompatibility of β-TCP has demonstrated its potential for applications in regenerative medicine due to its physicochemical characteristics, post-sintered mechanical properties, in vitro and in vivo performance, in addition to having Food and Drug Administration (FDA) approval for clinical use. Moreover, the possibility of extruding this material through a 3D printer has further expanded its potential for use in field of BTE. However, the absence of other mono- or poly-atomic ions like sodium (Na), magnesium (Mg), zinc (Zn), potassium (K), fluoride (F), chloride (Cl), and carbonate (CO $_{3}^{2-}$ ), all of which are known to play a vital role in bone metabolism and mineralization, has prompted the investigation of novel, ion-substituted bioceramics [44]. SBM is a carbonate hydroxyapatite containing Zn and Mg, which are known to dampen osteoclastic resorption and enhance osteoblast differentiation and maturation [45–49]. Furthermore, Mg has been suggested to promote osteoblastic motility and osteointegration of implantable devices at the bone-device interface [47–49].

A previous study had successfully demonstrated that SBM colloidal gel exhibited 3D printability and similar mechanical properties to β-TCP at comparable sintering temperatures [35]. For example, SBM 900 °C and β-TCP 1100 °C were found to exhibit similar flexural moduli [35]. However, cellular response to direct exposure of 3D printed SBM scaffolds remains unknown. In the present study, 3D printed scaffolds fabricated with SBM (sintered to 600 °C, 700 °C, or 1100 °C) and β-TCP (sintered to 1100 °C) were placed in direct contact with hOP cells. Both qualitative and quantitative evaluation of hOP cell cytotoxicity and proliferation, respectively, demonstrated inferior performance of SBM, relative to β-TCP at lower sintering temperatures. At higher sintering temperatures (1100 °C), qualitative cytotoxicity testing suggested that β-TCP presented no evidence of morphological change, while SBM 1100 °C showed few instances of acute cellular stress. Similar trends were realized in the quantitative cellular viability evaluation. Specifically, SBM 1100 °C yielded higher degrees of cell proliferation compared to SBM 600 °C after 168 h. Yet, at higher sintering temperatures (1100 °C), SBM still exhibited a significantly lower cell viability compared to β-TCP 1100 °C.

To determine the cause of these differences, the chemical changes that occur within SBM at varying sintering temperatures must be understood. It is known that carbonate content of SBM scaffolds decreases as a function of sintering temperature with near complete elimination at 800 °C and higher [35]. More specifically, the carbonate content of SBM at 600 °C, 700 °C, 800 °C, and 900 °C were found to be approximately 4.6%, 2.8%, 0.1% and 0.1%, respectively [35]. Therefore, SBM 1100 °C was the only group amongst SBM 600 °C, 700 °C, and 1100 °C to lack carbonate content. Yet, it demonstrated better in vitro performance relative to SBM scaffolds sintered at a lower temperature of 600 °C. It has been suggested that slow sintering between 600 °C and 700 °C, such as in the present study, results in decomposition of carbonate content with a resulting hinderance to cell proliferation [50,51]. This may be a plausible explanation for the limited cell proliferation within the SBM 600 °C group in comparison to SBM 700 °C and 1100 °C at 168 h. It is important to note that carbonate decomposition is said to occur to a lesser extent if sintered for a shorter duration, between 10–120 min, but not without compromise to mechanical strength [35,50,51]. However, the relationship between elemental composition of SBM and sintering temperature has yet to be established. As such, conclusive proof of this phenomenon through advanced techniques such as Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is warranted in future studies at the various sintering temperatures. This would allow for the quantification of ions present within media to better understand the primary cause of enhanced proliferative performance of cells seeded on SBM 1100 °C [52].

This pilot study examined the in vitro behavior of SBM sintered to 600 °C and 700 °C as these temperatures were previously reported to preserve SBM’s unique carbonate content [35]. Moreover, 1100 °C (well-documented as the ideal sintering temperature for bioceramics such as β-TCP) was selected to directly compare proliferative performance of SBM and β-TCP scaffolds [24–27]. Future studies are warranted and would benefit from an examination of cell cytotoxicity, proliferation, and mechanical strength of SBM at a broader range of sintering temperatures. Furthermore, the degradation profile of SBM relative to β-TCP must be determined in anticipation for use in vivo for hard tissue defect placement in follow-up studies.

5. Conclusion

SBM scaffolds seeded with hOP cells demonstrated inferior proliferative performance compared to β-TCP scaffolds. Lower sintering temperatures of SBM (600 °C and 700 °C) resulted in a higher degree of acute cellular stress relative to SBM 1100 °C. Thus, sintering temperatures of 600 °C and 700 °C are not advisable for post-processing of SBM scaffolds intended for bone regeneration. Although SBM still exhibited a significantly lower cell viability compared to β-TCP 1100 °C at higher sintering temperatures (1100 °C), minimal cellular stress was observed. Thus, SBM scaffolds sintered to 1100 °C may serve as a viable alternative to β-TCP scaffolds sintered at the same temperature.

Footnotes

Acknowledgements

The authors thank Dr. Bruce N. Cronstein’s lab (NYU Grossman School of Medicine) for the cell donation.

Author contributions

BVS - Analysis and Writing; NAM - Analysis and Writing; ZMS - Analysis and Writing; VVN - Investigation, Project administration, Supervision; Writing review and editing; JES - Methodology, and Investigation; CFR - Formal analysis, Methodology, Investigation; DQM - Methodology, Formal analysis, and Investigation; PGC - Writing review and editing; BNC - Writing review and editing; NT - Analysis and Writing; LW - Conceptualization, Formal analysis, Methodology, Investigation, Project administration, Resources, Supervision, and Writing review and editing. All authors have read and agreed to the published version of the manuscript.

Conflict of interest

The authors have no conflicts of interest to disclose.

Ethics statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of NYU (protocol code #S18-01579).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

The authors received no funding for the work.

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