At the beginning the main focus of three dimensional printing (3DP) was advanced manufacturing technology process, which was an appropriate for direct production in workshop or factory. Currently, three dimensional printing (3DP) is the most significant technology allows straight manufacturing in critical shape and structure with excellent resolution, in addition to processing decidedly customized healthcare products merged with image reconstitution method [1]. The progression of three dimensional printing (3DP) technologies has supplied doctors and researchers rich tools to advance the useful scaffolds that has achieved the firm condition in tissue engineering. The principle function of three dimensional printing (3DP) in tissue engineering is to provide the appropriate cell microenvironment for differentiation and proliferation of cells to the active tissue [2]. The two important methods of three dimensional printing (3DP) applying for tissue engineering at present. The first one is to fabricate scaffolds that can be cell cultured by in-vitro cells after the scaffold fabrication [3-4]. Another is constructing scaffolds in which cells are already present in the bioink. An additional is Polymer materials, which is also associated with the healthcare industry [5]. Different types of development of biodegradable polymers have been, as well as natural polymer and synthetic materials. The cons of the synthetic polymers that succeed over the natural polymers are that these synthetic type of polymers can alter their primary mechanical characteristics and also they have a plentiful resource of the fresh materials. Saturated aliphatic polyesters, for example poly (glycolic acid) (PGA), poly (lactic acid) (PLA), polycaprolactone (PCL), or their copolymers, are most generally applied in  tissue    materials, in addition to, it can be applied as three dimensional printing (3DP) materials [6].  Additionally, polymeric compounds that doped with reinforcement materials, for example carbon fibers or bioactive ceramics, are permitted to be processed by three dimensional printing (3DP) [7, 8]. The integration of bioactive hard stage into the polymers not it improves the mechanical characteristics of the printed scaffolds but it also improves the biological actions [9]. Along with the several issues need to be taken into consideration, mechanical characteristics of the printed scaffolds have be modified with respect to the exact position in the tissue of the host . Such as, the important compressive strength of scaffolds applied for cortical bone tissue is entirely dissimilar with that for a cancellous bone tissue engineering. For this purpose of bone segmental flaws of the cortical bone, scaffolds need compressive strength similar to its original model and also ranging from 100 to 150 MPa along with the axial track [10-11]. On the contrary, cancellous bone has a relatively slacken structure, ranging from 2.5–6.5 MPa [12]. Another mechanical characteristic, like as fracture toughness, elastic stiffness, and relaxation rate have to be controlled to remain reliable with original tissue [13-14]. Because the mechanical characteristic does not matches between the host tissue and scaffolds, which can cause stress defending [15], which ultimately effects in osteoporosis.

Alginate/Honey Bioink preparation

Sodium alginate was purchased form Sigma-Aldrich (Lot # SLBQ3067V) and honey (Dabur, India) was purchased from stationary shop. One solution of alginate/honey was prepared keeping the concentration of the alginate constant. 5% (w/v) Alginate and 5% Alginate with 0.5 % (w/v) Honey. Type III water (Wasserlab, Spain) was used for making the alginate solution then the concentration of honey was mixed in the solution. The solutions was stirred freshly by mechanical stirrer for 1 day for performing the 3D bioprinting and the experiment.

Physicochemical Characterization of Honay/Alginate

Physicochemical properties of the bioink like the pH, conductivity and viscosity were measured. The conductivity and the pH were measured at room temperature 27°C by using Thermo Oakton meter. Lamy Rheology Viscometer was used for measuring the viscosity of the solutions at room temperature 27°C by using spindle L-4 with torque 6 rpm and time 1 minute. 

Extrusion Based Bioprinter

 The 3D extrusion based bioprinter used for the experiment equipped with 4-axis movement system. X, Y, Z axis along with a syringe pump. The X and the Y axis are controlled by the stepper motors and the Z axis in controlled by a lead screw. Stepper motor with geared type is used to control the syringe pump for extrusion pressure and torque. The bed temperature can be controlled as per the requirement. SAM -32 bit controller with the printer software provides high accuracy and precision of the printed scaffolds. CURA an open source software used for slicing the solid model and for converting the .stl file to .GCODE format for uploading in the printer for printing. 

Figure 1: A) Input Structure Designed By Cura 15.04.4 Software, B) Output Printed Scaffolds Optical Images A) A5, B) A5H0.5

Scafflods Design

Sketchup 2017 an open source software which was used for designing the square  solid object of dimension X, Y, Z (34 × 34 × 0.7) mm and the saved file is in .stl format. CURA an open source software version 15.04.4 was used for slicing up the square solid object and making it into mesh like structure, by decreasing the parameter i.e. the fill density from 100 % to 17 % and the file is saved into .GCODE format. The .GCODE file format is uploaded in the printed software which is used for printing the scaffold. Fig. 1a represents the mesh like structure created by the software CURA.

Alginate/Honey Extrusion Printing

The two solutions Alginate/Honey and only the Alginate solutions were loaded into the 5ml syringe of the 3D bioprinter separately for printing. Printing speed was 50mm/s with nozzle diameter 0.4 mm at a room temperature 27°C. The solid mesh construct was printed very preciously by point-to-point method with a dimension of 34 × 34 × 5 mm. Crosslinking with CaCl₂  0.5 M was used postprinting for for keeping the desired structure of the scaffold stable. The printed scaffolds were printed on the glass Petridish which already contained the 0.5M CaCl₂ solution. The printed scaffolds were washed with 70% (Himedia, India) ethanol and vacuum dried at 40°C Figure 1b represents the optical images output scaffolds bioprinted constructs. 

Mechanical Characterization

Tensile testing was performed on the printed scaffolds by using UTM (Universal Testing Machine, Tenius Olson 5KT, and UK). The sample dimensions were taken 34 × 34 × 5 mm respectively. The tensile speed was taken 0.1 mm/min with fixed load cell of capacity 25 N. Clip-type sample holder was used for holding the samples. Triplicate sample testing was done on the samples.

Honey Release from the Printed Scaffolds

For measuring the honey release from the scaffold the scaffold A5H0.5 of dimension 1.5 × 1.5 cm were kept in PBS (Phosphate Buffer Solution) 10 mL and the supernatant release in the solution was measured at 2 and 4 days by using glucometer.  The other sample without honey was used as the blank. DPPH 2, 2-diphenyl-1-picrylhydrazyl antioxidant activity was measured by using sample A5H0.5 with A5 as blank.

Biological Study

For cell culture study scaffolds of dimension (10 × 10 mm × 100 µm) were used for studying the fibroblast 3T3 proliferation and adhesion. In 10% DMEM (Gibco, California) fetal bovine serum-supplemented Dulbecco’s modified Eagle’s medium solution containing 1% (penicillin–streptomycin) antibiotic the cells were cultured. From T25 flast calls were collected by trypsinization and centrifugation at for 5 min at 1500 rpm was done for getting resuspension in DMEM complete medium and for getting cell concentration of 2 × 10⁶ cells per mL. Cell seeding was further done on pure scaffolds of cell concentration of 10⁴ cells per mL and incubation was done at 5% CO₂ at temperature 37° C. On day 1 adhesion of the cells on the scaffolds the cell viability measurement was performed by 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in a 12 well plate. After cell seeding on the printed constructs cell trypsinization was done and after that MTT reagent was added on the scaffolds and incubation was done for 4 hours. In buffer the solution the formazan crystals which were formed are solubilized then the materials were moved to the 96 well plate. By using Multi Scan Go spectrometer (Thermo Fisher, Finland) the absorbance was measured at wavelength of 595 nm further the cellular viability of the scaffolds were also calculated. Same protocol was performed on day 3 for measuring the cellular viability. Cell viability was measured by the measured value recorded from the wavelength 595nm. Cell viability were performed on triplicate on each samples.

Alginate/honey Physicochemical Properties

In tissue engineering and drug discovery extrusion bioprinter have found lots of importance. Biomaterials fabrication with better cell-material and cell-cell interactions have been possible because of 3D bioprinting. Among the other bioprinter uused like laser based, inkjet based and extrusion based, extrusion based bioprinters are used mainly for fabrication of various biological constructs which are used in tissue engineering fields. The foremost challenges is to create an appropriate bioink which can provide suitable pH for the cells provided in them, shear thinning property must be present in the scaffolds so that it can be extruded from the nozzle without clogging and easily extruded as the low pressure is applied on them, the bioink should be such that it can sustain its structure and shape as rapidly as it is extruded from the printer nozzle it should solidify easily as soon as it extrudes from the nozzle of the extrusion 3D bioprinter. Crosslinking of the extruded scaffolds are also done for solidification, less damage is caused to the cells and maintaining the structural stability of the printed scaffolds. The cell adhesion and biocompatibility of the bioink should be high so that it cell-material interaction should be more compared to the cell-material interaction so that the cell can spread on the material easily and differentiation and proliferation of the cell can be increased. Till now alginate concentration of 2 to 4% was reported for fabrication of bioprinted scaffolds here in this paper we used alginate 5% mixed with low concentration of honey 0.5% and investigated the various physicochemical properties of the bioink like pH, conductivity and the viscosity. The viscosity of the bioinks were measured and founf out to be 9.1±0.5 and 7.7±0.3 Pa s of the two samples A5 and A5H0.5 respectively. For good printing the viscosity of the bioinks must be present between 0.3 – 30 Pa s. Here both the samples the viscosity is within this range. The viscosity of the solution containing honey is decreased because the viscosity of the honey is less compared to pure alginate. The honey components are getting inserted into the polymer chains of the alginate causing chain entanglement which in turns ultimately causes decrease is viscosity of the solution. The pH and conductivity of the solutions with honey were also reduces by incorporating honey with alginate solution. The pH of the two samples A5 and A5H0.5 are 7.5 and 7.4 respectively and conductivity of A5 and A5H0.5 are 28± 4.3 and 25±2.9 respectively. The cell responsiveness and the erratic degradation of the alginate printed scaffolds can be improved by mixing honey and gelatin with the alginate solution. Gelatin RGD sequences helps in cell adhesion. Disadvantage of gleatin addition with alginate causes the gelation temperature shiftinf to 11°C in 2% alginate –gelatin blends it is not in the case of honey so mixing honey in low concentration with the alginate bioinks can improve the erratic degradation and the cell responsiveness of the scaffolds maintaining the physicochemical and the printed structure stable.Optimization of the honey with fixed concentration of the alginate can be made for skin tissue specific applications and help tissue-specific stem cells differentiation. In figure 2b the glucose release and antioxidant potential of the scaffolds are shown. Glucose release was shown to be increased from day 0 to day 4. Although glucose release at day 0 was not found. The values of the respective glucose at day 2 and 4 of the sample A5H0.5 was found to be 41 and 108 mg/dL respectively which represents as with incubation the glucose release was found to be increased. The antioxidant scavenging percentage was also found out to be increase from day 2 to day 4. 2.1 % and 5.2 % were the respective scavenging percentage of day 2 and day 4 of the sample A5H0.5 as shown in figure 2c. 

Figure 2: A) Ultimate Tensile Stress of the Two Samples, B) Glucose Release Study of Sample A5H0.5 at Day 2 and Day 4, C) DPPH Antioxident Study of the Same Scaffold on Day 2 and Day 4

Figure 3: Cell Viability of the Printed Scaffolds at Day 2 and Day 4

Biological Studies of the Printed Scaffolds 

The biocompatibility of the printed constructs were performed by using fibroblast cells. 

The values of the MTT cell viability were significantly different from the blank A5 sample for both day 1 and 3 as shown in figure 3 (p≤0.05). Honey incorporation with the alginate improves the cell-material interaction more than the cell-material interaction. The mechanism by which honey incorporation with alginate which increases the cell viability is still not known. In day 3 the cell viability was more compared to the blank A5.

There are many bioinks which are under development like GelMA, alginate, collagen, agarose, poly (ethylene glycol), hyaluronic acid and silk. An appropriate biofabrication window must be present in bioinks which should provide the cell compatibility and rheology of the used polymer.  For cell differentiation and proliferation low polymer-loaded bioinks are used while high concentration are used for mechanical and rheology of the bioink. The disadvantages of high loaded-polymer is that it causes cell death by high pressure shear induced.  Honey the naturally available polymer has properties which induces cell proliferation and adhesion because of anti-bacterial, anti-oxidant, proangiogenic attributes and anti-inflammatory properties. Mixing honey at low concentration with the main bioink does not affect the rheological and physiological properties of the bioink in much extent.

Here in this paper we studied and presented Alginate/Honey bioinks scaffolds which can be used for in situ skin tissue engineering applications.  Here we mixed 0.5 % Honey with 5% Alginate and compared it with 5% Alginate scaffolds. Mixing honey reduced the viscosity but did not affect the structural stability too much. A little decrease in mechanical strength is observed in alginate blended scaffolds which can be applicable for soft tissue engineering applications like skin tissue engineering. Optimization of the percentage of honey in the range 1 to 5% can provide a good scaffold with highest cellular properties.  Seeing the benefits of honey as a possible wound healing means, this method may be a good technique for finding bioprinted scaffolds in the health center.

1. Barua, R. et al. Scaffold and Tissue Engineering Applications by 3D Bio-Printing Process: A New Approach. IGI Global, January 2019, pp. 78–99.

2. Derby, B. “Printing and Prototyping of Tissues and Scaffolds.” Science, vol. 338, no. 6109, 2012, pp. 921–926.

3. Altamimi, A.A. et al. “Metallic Bone Fixation Implants: A Novel Design Approach for Reducing the Stress Shielding Phenomenon.” Virtual and Physical Prototyping, vol. 12, no. 2, 2017, pp. 141–151.

4. Zhang, Y. et al. “Custom Prosthetic Reconstruction for Proximal Tibial Osteosarcoma with Proximal Tibiofibular Joint Involved.” Surgical Oncology, vol. 17, no. 2, 2008, pp. 87–95.

5. Datta, S. et al. “Alginate-Honey Bioinks with Improved Cell Responses for Applications as Bioprinted Tissue Engineered Constructs.” July 2018.

6. Datta, S. et al. “Design and Development of Alginate: Poly-L-Lysine Scaffolds by 3D Bioprinting and Studying Their Mechanical, Structural and Cell Viability.” September 2018.

7. Datta, S. et al. “Alginate-Poly(Amino Acid) Extrusion Printed Scaffolds for Tissue Engineering Applications.” International Journal of Polymeric Materials and Polymeric Biomaterials, vol. 69, no. 2, 2018, pp. 1–9.

8. Datta, S. et al. “Bioink Formulations to Ameliorate Bioprinting-Induced Loss of Cellular Viability.” AVS, vol. 14, no. 5, September 2019.

9. Barua, R. et al. “Importance of 3D Printing Technology in Medical Fields.” IGI Global, January 2019.

10. Hutmacher, D.W. “Scaffolds in Tissue Engineering Bone and Cartilage.” Biomaterials, vol. 21, no. 24, 2000, pp. 2529–2543.

11. Pan, T. and X. Cao. “Progress in the Development of Hydrogel-Rapid Prototyping for Tissue Engineering.” Materials China, vol. 34, no. 3, 2015, pp. 236–245.

12. Ozbolat, I.T. and M. Hospodiuk. “Current Advances and Future Perspectives in Extrusion-Based Bioprinting.” Biomaterials, vol. 76, no. 37, 2016, pp. 321–343.

13. Gudapati, H. et al. “A Comprehensive Review on Droplet-Based Bioprinting: Past, Present and Future.” Biomaterials, vol. 102, 2016, pp. 20–42.

14. Ng, W.L. et al. “Microvalve-Based Bioprinting—Process, Bio-Inks and Applications.” Biomaterial Science, vol. 5, no. 4, 2017, pp. 632–647.

15. Koch, L. et al. “Laser-Assisted Bioprinting at Different Wavelengths and Pulse Durations with a Metal Dynamic Release Layer: A Parametric Study.” International Journal of Bioprinting, vol. 3, no. 1, 2017, pp. 42–53.