Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

The scope of the research is focused on evaluating the potential of apple-derived cellulose scaffold for bone tissue engineering and assessing their mechanical properties both in vitro and in vivo. The question addressed include the osteogenic potentials of the scaffold, their ability to support cell invasion, mineralization, and their mechanical performance. The most recent development in the field of research involved the utilization of cellulose-based scaffold derived from various plants for tissue reconstruction.

These scaffolds can be shaped and modified to achieve desired characteristics, and functionalization techniques have been employed to enhance their effectiveness. The research gap addressed by this protocol is the limited investigation of the mechanical properties of apple-derived cellulose for bone tissue engineering. While previous studies have explored potential application of the scaffolds, their mechanical characteristics have not been extensively studied.

The present study fills the gap by evaluating the mechanical properties of the scaffolds, highlighting their limitation compared to healthy bone tissue, and emphasizing the need for further development to optimize their performance. The findings of this study contribute to the advancements of research in the field by confirming that cellulose scaffold their from apples have the potential to promote adhesion osteoblastic differentiation and proliferation of preosteoblastic cells. Following the study, one can ask questions such as, how can the mechanical properties of cellulose scaffolds that are from plants be optimized to closely match or mimic those of natural bone tissue?

Chemical modifications or composite strategy can be employed to enhance the stiffness and load-bearing capacity of plant-derived cellulose scaffolds for bone tissue engineering applications. To begin, use a mandoline slicers to cut Macintosh apples into eight millimeter thick slices. Cut the hypanthium tissue of the apple slices into 5-by-5 millimeter squares.

Place the square samples in 0.1%SDS for two days to decellularize them. After incubation, wash the samples with deionized water and incubate them overnight in 100 millimolar calcium chloride at room temperature, then sterilize these scaffolds in 70%ethanol for 30 minutes. Wash them with deionized water and place them in a 24 well culture plate.

For cell seeding, use MC3T3-E1 subclone 4 cells maintained in 10 centimeter diameter cell culture treated dishes in cell culture conditions. Also prepare a cell culture medium composed of Alpha MEM supplemented with 10%fetal bovine serum or FBS and 1%penicillin in streptomycin. Once the cells reach 80%confluency, detach them from the culture dishes by trypsinization.

Centrifuge the cell suspension at 200g for three minutes. Aspirate the supernatant and resuspend the cells in Alpha MEM. Next, pipette 40 microliter aliquot of the cell suspension on the surface of the scaffolds and let the cells adhere for one hour in cell culture conditions.

Subsequently, add two milliliters of culture medium to each culture well. Replenish the culture medium every two to three days for 14 days. After 14 days, prepare differentiation medium by adding 50 micrograms per milliliter of ascorbic acid and four millimolar sodium phosphate to the cell culture medium.

Add this medium to the culture plate. Incubate the scaffolds for four weeks to induce differentiation of MC3T3-E1 cells, and replenish the medium every three to four days. To begin, wash the decellularized apple scaffolds with phosphate buffered saline.

For pore size measurements, incubate the scaffolds in one milliliter of 10%Calcofluor White solution for 25 minutes in the dark at room temperature. After washing the scaffolds with PBS, use a high speed resonant confocal laser scanning microscope and set the laser emission filter configuration. Adjust the laser power and detector manually to ensure optimal image acquisition.

Acquire a Z-stack of 20 images with a five micrometer step size. Next, in the ImageJ software, use the Z Project to maximum intensity function to create an image and apply the Find Edges function to highlight the edge of the pores. Trace the pores manually using the Freehand Selection tool.

Fit each pore as knee lips and measure the length of the major axis. Compile all the measurements and calculate the average length. For the analysis of cell distribution, after washing the cell-seeded scaffolds cultured in the appropriate medium three times with PBS, fix them with 4%paraformaldehyde for 10 minutes.

Then thoroughly wash each scaffold with deionized water, permeabilize the cells with a Triton X-100 solution for five minutes and wash again with PBS. Incubate the scaffolds in one milliliter of 1%periodic acid for 40 minutes and rinse with deionized water. Then incubate the scaffolds by completely immersing them in one milliliter of staining solution.

Wash the scaffolds with PBS and stain the cell nuclei by incubating the scaffolds in a five milligrams per milliliter DAPI solution for 10 minutes in the dark. Thoroughly wash again and store the scaffolds in PBS. Image the cell-seeded scaffolds with a high speed resonant confocal laser scanning microscope at 10x magnification using the settings shown here.

Adjust the laser power and detector manually to ensure optimal image acquisition and acquire a Z-stack of 20 images with a five micrometer step size. Then use ImageJ software to process the confocal images and use the Z Project to maximum intensity function to create a maximum projection in the Z-axis for image analysis. The quantification of the confocal microscopy images demonstrated a pore size distribution between 73 to 288 micrometers with an average of around 154 micrometers.

The majority of the pores ranged between 100 and 200 micrometers. After culturing in the differentiation medium, mineral deposits were observed in the scaffolds. The cell-seeded scaffolds displayed an opaque white coloration suggesting mineralization, which was not observed in the blank scaffolds.

Furthermore, confocal laser scanning microscopy analysis revealed a homogenous cell distribution within the scaffolds. For the alkaline phosphatase assay and calcium deposition assays begin by washing the cell-seeded apple-derived scaffolds cultured in the appropriate medium three times with PBS. Fix the scaffolds with 10%neutral buffered formalin for 30 minutes.

For alkaline phosphatase assay, prepare BCIP/NBT staining solution by dissolving one BCIP/NBT tablet in 10 milliliters of deionized water. Then wash the fixed scaffolds with a 0.05%Tween solution and stain with the BCIP/NBT solution for 20 minutes at room temperature. Wash the stained scaffolds with a 0.05%Tween solution and store them in PBS.

Then image the stain scaffolds with a 12 megapixel digital camera. For calcium deposition assay, prepare a 2%weight by volume Alizarin Red S staining solution. Then wash the fixed scaffolds with deionized water and stain them with the Alizarin Red S solution for one hour at room temperature.

Wash the stained scaffolds with deionized water and store them in PBS prior to imaging. Image with a 12 megapixel digital camera. To perform Young's modulus measurements, remove the cell-seeded scaffolds from their respective incubation medium and immediately test the samples using a custom built uniaxial compression apparatus equipped with a 1.5 Newton load cell.

Compress the scaffolds at a constant rate of three millimeters per minute to a maximum compressive strain of 10%of the scaffold height. Determine Young's modulus from the slope of the linear portion of the stress strain curves. BCIP/NBT staining revealed a substantial increase in alkaline phosphatase activity in the cell-seeded scaffolds cultured in the differentiation medium compared to the blank or non-differentiating medium culture cell-seeded scaffolds.

Similar results were obtained for Alizarin Red S staining indicating greater calcium mineralization in the cell-seeded scaffolds cultured in the differentiation medium. The Young's modulus of the cell-seeded scaffolds cultured in differentiation medium was around 192.0 kilopascal, which was significantly higher than the blank or non-differentiation medium culture cell-seeded scaffolds indicating higher stiffness.