Graphical abstract
Highlights
- •
Stearic acid/tricalcium phosphate suspensions may be 3D printed with a rapid and simple process.
- •
Sintering results in a chemically pure and mechanically strong ceramics.
- •
Mesenchymal stem cells are able to adhere and form new bone on these ceramics.
Abstract
Objective
Craniofacial bone trauma is a leading reason for surgery at most hospitals. Large pieces of destroyed or resected bone are often replaced with non-resorbable and stock implants, and these are associated with a variety of problems. This paper explores the use of a novel fatty acid/calcium phosphate suspension melt for simple additive manufacturing of ceramic tricalcium phosphate implants.
Methods
A wide variety of non-aqueous liquids were tested to determine the formulation of a storable 3D printable tricalcium phosphate suspension ink, and only fatty acid-based inks were found to work. A heated stearic acid-tricalcium phosphate suspension melt was then 3D printed, carbonized and sintered, yielding implants with controllable macroporosities. Their microstructure, compressive strength and chemical purity were analyzed with electron microscopy, mechanical testing and Raman spectroscopy, respectively. Mesenchymal stem cell culture was used to assess their osteoconductivity as defined by collagen deposition, alkaline phosphatase secretion and de-novo mineralization.
Results
After a rapid sintering process, the implants retained their pre-sintering shape with open pores. They possessed clinically relevant mechanical strength and were chemically pure. They supported adhesion of mesenchymal stem cells, and these were able to deposit collagen onto the implants, secrete alkaline phosphatase and further mineralize the ceramic.
Significance
The tricalcium phosphate/fatty acid ink described here and its 3D printing may be sufficiently simple and effective to enable rapid, on-demand and in-hospital fabrication of individualized ceramic implants that allow clinicians to use them for treatment of bone trauma.
1
Introduction
Bone trauma is a common condition that is treated at most hospitals and which may occur due to a number of reasons such as accidents, falls, violence, surgery, infection, degenerative diseases and cancer resection . Some bone defects and fractures are treated with titanium mini-plates and screws as bone heals on its own. However, permanent implants may be required to replace pieces of resected or destroyed bone that are either too large to heal on their own or where the restoration of a particular anatomical feature is critical to the aesthetic or functional outcome . The current paper focuses on this type of implant. Autologous bone may be used for such implants but tissue harvesting results in increased morbidity and increased time of surgery. Implants made from non-resorbable materials are common alternatives, but, depending on the type of defect and type of implant, such materials are at risk of infection and rejection and may stress shield surrounding bone, generate wear debris and loosen over time. Furthermore, non-resorbable implants cannot be repaired and remodeled by the body and often need to be replaced in young growing patients. Degradable (resorbable) implant materials may solve these problems . Ceramic calcium phosphate-based implants are an established alternative to non-resorbable implant materials and autologous bone and have been used clinically for many years . Tricalcium phosphate (TCP) is especially interesting as it slowly biodegrades and is remodeled to true bone to which it contributes calcium and phosphate. To accelerate new bone formation, bone forming cells such as mesenchymal stem cells (MSCs) are often added to implants .
To further improve the use of resorbable, tricalcium-based implants, the use of three dimensional (3D) printing has evolved to a level at which such techniques can actually contribute to more predictable reconstructions. 3D printing, a method of additive manufacturing, is a computer-assisted manufacturing method where an object is built layer-by-layer using a digital object model also known as a CAD model. It has recently been applied to the fabrication of bone implants as it can be used to produce individualized implants that recapitulate patient anatomy especially when used in conjunction with patient scanning data and virtual surgical planning . 3D printing also enables the formation of bone-forming pores with specific diameters and direction within the implant. TCP can be 3D printed using different methods, including stereolithography , selective laser sintering , binder ink jetting , and robocasting. Robocasting, also known as direct writing and extrusion printing, is an additive manufacturing technique where an “ink” is deposited by a computer-controlled extruder onto a build platform . Robocasting is an attractive technique for in-hospital 3D printing due to its simplicity, low-cost, low-maintenance and its capability to use any material and combinations of several different materials in one print . Several previous studies have used robocasting to produce calcium phosphate implants that have successfully supported osteogenesis in vitro and in vivo . Recently, a number of simple-to-operate and low-cost (under $ 10,000) robocasting capable 3D printers have become commercially available; these include the Biobot’s Biobot , Cellink’s Inkredible , and Hyrel’s System 30M . The simplicity and pricing of these 3D printers could enable low-cost and on-demand 3D printing of individualized implants locally at hospitals. This also requires an ink that is equally low-cost and simple to prepare and handle.
Most publications concerning robocasting describe inks that are complex aqueous powder suspensions that contain multiple additives and which often take several hours to prepare. Aqueous suspension may also suffer from problems such as detrimental influence from their pH and uneven dehydration during printing. Strategies for improving robocasting such as using inks that gel independently of pH or by printing into an oil bath are therefore of great interest. However, all water-based prints must be fully dehydrated before sintering as the boiling water produced in the process would otherwise introduce cracks in the final ceramic. This dehydration step typically takes one or more days. Aqueous formulations may also support chemical reactions such as oxidation, hydration or recrystallization of the powder, resulting in a non-usable ink; this precludes extended storage and requires fresh inks to be prepared for each print. The need for fresh inks, the complexity and time required to prepare such inks and the extended post-printing dehydration time present a significant obstacle to rapid, local, on-demand printing of implants in hospitals.
Long aliphatic chains efficiently lubricate surfaces, and molecules that contain such chains are often used as lubricants. Stearic acid, for example, lowers inter-particle attraction and granular shear strength . This paper investigates the use of such lubricants to create a new calcium phosphate implant fabrication method that relies on a simple-to-prepare, two-component, non-aqueous robocasting ink. Our aim was to develop a simple and inexpensive individualized implant production method that could be implemented at hospitals without compromising implant strength, biocompatibility or osteoconductivity.
2
Methods
2.1
Materials
Tricalcium phosphate (TCP, Cat. No. 21218), ascorbic acid, dexamethasone, calcitriol, betaglycerol phosphate and stearic acid, copper powder and copper oxide were purchased from Sigma–Aldrich (St. Louis, MO, USA). MEM medium, penicillin/streptomycin (P/S), trypsin and fetal bovine serum (FBS) were acquired from Invitrogen (Waltham, MA, USA).
2.2
Ink testing
To replace water in the robocasting ink, TCP was combined at various ratios with a large number of potential lubricants including decane, oleic acid, oleyl alcohol, oleyl ester, glycerol, triglycerides (sunflower oil) and heated paraffin. The viscoelastic properties of the inks were tested qualitatively by stirring the suspensions with a spoon. Their extrusion/3D printing properties were tested by placing them in an aluminum syringe that was placed into a System 30 3D printer (Hyrel 3D, Atlanta, GA, USA). Using the printer’s manual settings, the syringe was then pressurized and the extruded liquid or suspension was visually and physically inspected.
2.3
Implant preparation
Inks for implant preparation were formulated by mixing 25 g TCP with 5 g stearic acid (W/W 83%, V/V 60%) and heating the mixture to 80 °C. This ink was loaded into an aluminum syringe that was placed into a heatable syringe extrusion head (a Vol-25, Hyrel 3D, Atlanta, GA, USA). CAD files of 50 mm × 50 mm × 3 mm or 20 mm × 20 mm × 20 mm boxes were constructed using Autodesk Inventor 2015 and exported as STL files. This files were imported into a System 30 3D printer, where they were prepared for printing using Slic3r 1.2.9. Settings were a layer height of 0.2 mm, a print speed of 15 mm/s, an infill of 70%, a rectangular infill pattern, a solid top and bottom value of 0 and a perimeter value of 0. The extrusion head was heated to 80 °C and remained at this temperature during the entire print. The printing bed was heated to 40 °C for the first layer of the print to facilitate ink adhesion but was not heated for the remaining prints where the approximate bed temperature was 25 °C. Printing was done through a 1 mm nozzle. After printing, the 50 mm × 50 mm × 3 mm print was carefully removed from the printing bed and was cut into smaller 7 mm × 7 mm × 3 mm implants containing four pores. The smaller implants were not printed directly as the 3D printer software used at the time of the study had difficulty printing the edges of small objects, by printing and trimming larger objects this problem could be eliminated. This is not a problem for clinical implementability as defects that small would self-heal anyway. The larger 20 mm × 20 mm × 20 mm implants, a clinically relevant size, were 3D printed directly. Except where noted, all implants were placed into a pre-heated oven (Nabertherm L3) for 1 h at 400 °C and were then sintered for 2 h at 1100 °C followed by slow cooling to room temperature (1–2 h). All experiments were conducted with the 7 mm × 7 mm × 3 mm implants, except for the mechanical testing.
2.4
Cell seeding and differentiation
For the initial experiment three scaffolds were placed in each well on ultra-low-adherence 24-well plates (Corning) that was then added 100,000 cells eGFP + hMSC (Tert4 + , p62) cells in 500 μL maintenance medium (MEM medium with 1% P/S and 10% FBS). For the remaining experiments with eGFP − hMSCs, single implants were placed in clean ultra-low-adherence 24-well plates to which was added 200,000 eGFP − hMSCs (Tert4, p45) in 50 μL maintenance medium. After 30 min, 1 mL medium was added to each well. After 48 h, the medium was replaced with either 1 mL maintenance medium or osteogenic medium (maintenance medium plus 10 mM betaglycerol phosphate, 10 nM dexamethasone, 10 nM calcitriol and 250 nM ascorbic acid). Medium was then changed twice weekly. The day number 2 + X refers to the 2 days of culture in maintenance medium and X days in either osteogenic or maintenance medium. Implants were visualized using an inverted phase contrast microscope (Olympus IX50) or an inverted epifluorescence microscope (Leica), both at ×10 magnification. Representative images are shown.
2.5
Raman spectroscopy of implants
The implant material was investigated for unwanted chemical reactions and contaminations at various stages using Raman spectroscopy. Raman spectra were obtained using an in-house build Raman microscope. For laser excitation a Laser Quantum Ventus 532 nm Laser (Stockport, UK). The laser is coupled via free space optics into a Olympus BX60 microscope with a 50× objective (Hamburg, Germany) that are fiber-coupled to an Acton SpectraPro 2500i f/6.5 spectrograph, using a 600 L/mm grating, blazed at 500 nm and with a Princeton instruments PIXIS 400F 1340 × 400 pixel CCD camera (Trenton, NJ, USA) operating at −75 °C.
For stearic acid 10 mW of laser power was applied to the sample and for TCP and implants 30 mW was applied. Integration times was 10 s with 3–10 averages to achieve a comparable SNR. The spectra were offset-corrected and normalized to the maximum value.
2.6
Scanning electron microscopy of implants
Implant microstructure was investigated with scanning electron microscopy (SEM). Samples were prepared for inspection in a SEM (JSM 6480, JEOL), the implant with cells were washed 3 times in distilled water, fixed in 3.7% formaldehyde for 10 min at room temperature, dehydrated by air drying, and finally coated with 10 nm of gold in a Cryofox thermal evaporater (Polyteknik A/S, Østervrå, Denmark). The scanning electron microscope (SEM) images were recorded at acceleration voltages of 10 kV and 20 kV and working distance of 15 mm.
2.7
Mechanical testing of implant
The mechanical properties of the implants where determined by compression testing in a Zwick Z050 universal testing machine (Zwick Roell, Ulm, Germany). The measured values were: Maximum stress − σ max [N/mm 2 ], strain at σ max − dL [%] and Youngs Modulus − E [MPa]. The test was performed position-controlled with a test of speed 1 mm/s during the entire test sequence.
2.8
Investigating implant osteoconductivity with cell titer and alkaline phosphatase activity
After 2 + 7 days of cell culture, the implants were transferred to wells on a 48 well plate to exclude any cells residing on bottom of the culture well from analysis; to the implants were added 40 μL CellTiter and 200 μL Maintenance Medium. After 30 min, 3 × 70 μL medium was transferred from each well to a black 96 well plate. Viability was measured as fluorescence according to the manufacturer’s protocol using a FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany). After determination of viability, the same implants were washed 3 times with PBS, rinsed with TBS, fixed for 30 s in 90% Ethanol with 3.7% formaldehyde, the fixative was then removed and the implants were incubated with 250 μL 1 mg/mL 4-nitrophenol phosphate for 20 min. The reaction was then stopped with 150 μL 3 M NaOH. 3 × 100 μL from each well was then transferred to a clear 96 well plate. Alkaline phosphatase activity was measured as absorbance using a FLUOstar OPTIMA. For each well the average alkaline phosphatase activity was divided by the average viability value to provide a cell number normalized alkaline phosphatase activity measure.
2.9
Investigating implant osteoconductivity with sirius red and fast green staining
After 2 + 7 and 2 + 25 days of cell culture, the implants were transferred to wells on a 48 well plate to exclude any cells residing on bottom of the culture well from analysis. The implants were then washed two times with PBS and were then fixed in Kahle’s fixative (3.7% formaldehyde, 1% acetic acid, 26.88% EtOH) for 10 min at room temperature. The implants were then washed twice with PBS and was then stored at 4 °C in 1 mL PBS until staining. For staining a sirius red/fast green staining kit was used (Chondrex, Redmond, WA, USA), the PBS was removed and 300 μL dye solution was added to each well, after 30 min at room temperature the solutions was removed and the wells were washed with 1 mL water until the water was clear (15 times). The wells were photographed and 1 mL of extraction buffer added; after 20-min incubation on a rocking table at room temperature 3 × 200 μL from each well was transferred to a 96-well plate and absorbance was read at 540 nm and 605 nm. The collagen and non-collagenous protein content was then determined using the following equations from the kit manufacturer’s protocol:
2.10
Investigating implant osteoconductivity with micro-computed tomography scanning
The same implants were scanned before and after 2 + 25 days of cell culture. Implants with cells were washed three times in distilled water, fixed in 3.7% formaldehyde for 10 min at room temperature, washed three times in distilled water and dehydrated by air drying before being scanned. The implants were wrapped in plastic film and placed in one scanning cylinder that was scanned using a Scanco vivaCT40 using the same settings before and after cell culture. The resulting ISQ files were imported into ImageJ (1.49v) using the KHKs microCT Tools add on and with the following settings: “Downsample by factor 2 in x, y, z (method = average)” and “8-bit-import”. Five slices were then selected at the center of each implant but with a minimum spacing of 10 slices. In each of these slices, the implant was selected as the region of interest, a histogram was generated and the values exported. An identical intensity threshold was then applied to all data sets to analyze only bone; bone density was then found as: Bone Density = Intensity Level Value(x) * Bone Pixels with Intensity Value (x)/Total Bone Pixels.
2.11
Statistics
Figs. 6 and 7 show mean values with sample standard deviation on the error bars. The number of biological replicates is indicated as n in each figure text. Two-tailed t-Tests assuming unequal variance were performed to check for difference between mean values; p < 0.05 was taken to indicate a statistically significant difference.
2
Methods
2.1
Materials
Tricalcium phosphate (TCP, Cat. No. 21218), ascorbic acid, dexamethasone, calcitriol, betaglycerol phosphate and stearic acid, copper powder and copper oxide were purchased from Sigma–Aldrich (St. Louis, MO, USA). MEM medium, penicillin/streptomycin (P/S), trypsin and fetal bovine serum (FBS) were acquired from Invitrogen (Waltham, MA, USA).
2.2
Ink testing
To replace water in the robocasting ink, TCP was combined at various ratios with a large number of potential lubricants including decane, oleic acid, oleyl alcohol, oleyl ester, glycerol, triglycerides (sunflower oil) and heated paraffin. The viscoelastic properties of the inks were tested qualitatively by stirring the suspensions with a spoon. Their extrusion/3D printing properties were tested by placing them in an aluminum syringe that was placed into a System 30 3D printer (Hyrel 3D, Atlanta, GA, USA). Using the printer’s manual settings, the syringe was then pressurized and the extruded liquid or suspension was visually and physically inspected.
2.3
Implant preparation
Inks for implant preparation were formulated by mixing 25 g TCP with 5 g stearic acid (W/W 83%, V/V 60%) and heating the mixture to 80 °C. This ink was loaded into an aluminum syringe that was placed into a heatable syringe extrusion head (a Vol-25, Hyrel 3D, Atlanta, GA, USA). CAD files of 50 mm × 50 mm × 3 mm or 20 mm × 20 mm × 20 mm boxes were constructed using Autodesk Inventor 2015 and exported as STL files. This files were imported into a System 30 3D printer, where they were prepared for printing using Slic3r 1.2.9. Settings were a layer height of 0.2 mm, a print speed of 15 mm/s, an infill of 70%, a rectangular infill pattern, a solid top and bottom value of 0 and a perimeter value of 0. The extrusion head was heated to 80 °C and remained at this temperature during the entire print. The printing bed was heated to 40 °C for the first layer of the print to facilitate ink adhesion but was not heated for the remaining prints where the approximate bed temperature was 25 °C. Printing was done through a 1 mm nozzle. After printing, the 50 mm × 50 mm × 3 mm print was carefully removed from the printing bed and was cut into smaller 7 mm × 7 mm × 3 mm implants containing four pores. The smaller implants were not printed directly as the 3D printer software used at the time of the study had difficulty printing the edges of small objects, by printing and trimming larger objects this problem could be eliminated. This is not a problem for clinical implementability as defects that small would self-heal anyway. The larger 20 mm × 20 mm × 20 mm implants, a clinically relevant size, were 3D printed directly. Except where noted, all implants were placed into a pre-heated oven (Nabertherm L3) for 1 h at 400 °C and were then sintered for 2 h at 1100 °C followed by slow cooling to room temperature (1–2 h). All experiments were conducted with the 7 mm × 7 mm × 3 mm implants, except for the mechanical testing.
2.4
Cell seeding and differentiation
For the initial experiment three scaffolds were placed in each well on ultra-low-adherence 24-well plates (Corning) that was then added 100,000 cells eGFP + hMSC (Tert4 + , p62) cells in 500 μL maintenance medium (MEM medium with 1% P/S and 10% FBS). For the remaining experiments with eGFP − hMSCs, single implants were placed in clean ultra-low-adherence 24-well plates to which was added 200,000 eGFP − hMSCs (Tert4, p45) in 50 μL maintenance medium. After 30 min, 1 mL medium was added to each well. After 48 h, the medium was replaced with either 1 mL maintenance medium or osteogenic medium (maintenance medium plus 10 mM betaglycerol phosphate, 10 nM dexamethasone, 10 nM calcitriol and 250 nM ascorbic acid). Medium was then changed twice weekly. The day number 2 + X refers to the 2 days of culture in maintenance medium and X days in either osteogenic or maintenance medium. Implants were visualized using an inverted phase contrast microscope (Olympus IX50) or an inverted epifluorescence microscope (Leica), both at ×10 magnification. Representative images are shown.
2.5
Raman spectroscopy of implants
The implant material was investigated for unwanted chemical reactions and contaminations at various stages using Raman spectroscopy. Raman spectra were obtained using an in-house build Raman microscope. For laser excitation a Laser Quantum Ventus 532 nm Laser (Stockport, UK). The laser is coupled via free space optics into a Olympus BX60 microscope with a 50× objective (Hamburg, Germany) that are fiber-coupled to an Acton SpectraPro 2500i f/6.5 spectrograph, using a 600 L/mm grating, blazed at 500 nm and with a Princeton instruments PIXIS 400F 1340 × 400 pixel CCD camera (Trenton, NJ, USA) operating at −75 °C.
For stearic acid 10 mW of laser power was applied to the sample and for TCP and implants 30 mW was applied. Integration times was 10 s with 3–10 averages to achieve a comparable SNR. The spectra were offset-corrected and normalized to the maximum value.
2.6
Scanning electron microscopy of implants
Implant microstructure was investigated with scanning electron microscopy (SEM). Samples were prepared for inspection in a SEM (JSM 6480, JEOL), the implant with cells were washed 3 times in distilled water, fixed in 3.7% formaldehyde for 10 min at room temperature, dehydrated by air drying, and finally coated with 10 nm of gold in a Cryofox thermal evaporater (Polyteknik A/S, Østervrå, Denmark). The scanning electron microscope (SEM) images were recorded at acceleration voltages of 10 kV and 20 kV and working distance of 15 mm.
2.7
Mechanical testing of implant
The mechanical properties of the implants where determined by compression testing in a Zwick Z050 universal testing machine (Zwick Roell, Ulm, Germany). The measured values were: Maximum stress − σ max [N/mm 2 ], strain at σ max − dL [%] and Youngs Modulus − E [MPa]. The test was performed position-controlled with a test of speed 1 mm/s during the entire test sequence.
2.8
Investigating implant osteoconductivity with cell titer and alkaline phosphatase activity
After 2 + 7 days of cell culture, the implants were transferred to wells on a 48 well plate to exclude any cells residing on bottom of the culture well from analysis; to the implants were added 40 μL CellTiter and 200 μL Maintenance Medium. After 30 min, 3 × 70 μL medium was transferred from each well to a black 96 well plate. Viability was measured as fluorescence according to the manufacturer’s protocol using a FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany). After determination of viability, the same implants were washed 3 times with PBS, rinsed with TBS, fixed for 30 s in 90% Ethanol with 3.7% formaldehyde, the fixative was then removed and the implants were incubated with 250 μL 1 mg/mL 4-nitrophenol phosphate for 20 min. The reaction was then stopped with 150 μL 3 M NaOH. 3 × 100 μL from each well was then transferred to a clear 96 well plate. Alkaline phosphatase activity was measured as absorbance using a FLUOstar OPTIMA. For each well the average alkaline phosphatase activity was divided by the average viability value to provide a cell number normalized alkaline phosphatase activity measure.
2.9
Investigating implant osteoconductivity with sirius red and fast green staining
After 2 + 7 and 2 + 25 days of cell culture, the implants were transferred to wells on a 48 well plate to exclude any cells residing on bottom of the culture well from analysis. The implants were then washed two times with PBS and were then fixed in Kahle’s fixative (3.7% formaldehyde, 1% acetic acid, 26.88% EtOH) for 10 min at room temperature. The implants were then washed twice with PBS and was then stored at 4 °C in 1 mL PBS until staining. For staining a sirius red/fast green staining kit was used (Chondrex, Redmond, WA, USA), the PBS was removed and 300 μL dye solution was added to each well, after 30 min at room temperature the solutions was removed and the wells were washed with 1 mL water until the water was clear (15 times). The wells were photographed and 1 mL of extraction buffer added; after 20-min incubation on a rocking table at room temperature 3 × 200 μL from each well was transferred to a 96-well plate and absorbance was read at 540 nm and 605 nm. The collagen and non-collagenous protein content was then determined using the following equations from the kit manufacturer’s protocol:
Stay updated, free dental videos. Join our Telegram channel
VIDEdental - Online dental courses
