MEEN41410 Tissue Engineering Assignment 1 : Statistical Report 2026 | UCD

MEEN41410 Tissue Engineering Assignment 1

Module co-ordinator: Dr Fiona Freeman

Overall Assessment: 20% of final grade

Assignment Description

In this assignment, you will be assigned a specific dataset (refer to Brightspace for your assigned data). Half the class will focus on bone regeneration, while the other half will assess cartilage regeneration. Your task is to analyze the raw data from biochemical assays previously conducted. You will need to generate a standard curve and interpolate your data according to the biochemical assays. Based on your results, you will select appropriate statistical tests, perform the analyses using GraphPad, and present your findings in a lab report.

Specific Instructions

1) Biochemical Assays

• • Bone Regeneration Data Sets: Your dataset will include two assays: Calcium and Alkaline Phosphatase (ALP) assays.
  • Cartilage Regeneration Data Sets: Your dataset will include two assays: DNA and sulfated glycosaminoglycan (sGAG) assays.
  • You are required to generate standard curves for both assays and interpolate your experimental data using the appropriate equations.
  • Include your final interpolated data table in the lab report, along with the equation used for interpolation. Additionally, submit the Excel spreadsheet containing your equations.

2) Experiments

• • Bone Regeneration Study: This study evaluates the bone regenerative potential of 3D bioprinted scaffolds with or without the addition of decellularized bone extracellular matrix (ECM) and/or Bone Morphogenetic Protein 2 (BMP-2). The experimental groups are:
    1. 3DP Scaffold (no cells)
    2. 3DP Scaffold + bone ECM (no cells)
    3. 3DP Scaffold (with cells)
    4. 3DP Scaffold + bone ECM (with cells)
    5. 3DP Scaffold + BMP-2
    6. 3DP Scaffold + bone ECM + BMP-2

You will interpolate your assigned dataset for both Calcium and ALP assays across all experimental groups based on your dataset. For the Calcium assay we used 10uL of a 500ul sample within each well to conduct the assay and the following standards were used:

For the ALP Assay we used a 50uL of our sample and performed the ALP assay over 60 minutes. To interpolate your data, you need to use the following equation: ALP Activity (µmol/minute/mL) = A/V/T. Where A is amount of pNP generated by samples (in µM).  V is volume of sample added in the assay well (in mL).  T is reaction time (in minutes). The following standards were used:

• Cartilage Regeneration Study: This study evaluates the chondrogenic potential of a hyaluronic acid hydrogel scaffold cultured under normoxia or hypoxia, with or without transforming growth factor beta 3 (TGF-β3). The experimental groups are:
  1. HA Scaffold (no cells)
  2. HA Scaffold in Hypoxia (no cells)

• HA Scaffold (with cells)

1. HA Scaffold in Hypoxia (with cells)
2. HA Scaffold + TGF-β3
3. HA Scaffold in Hypoxia + TGF-β3

You will interpolate your assigned dataset for both DNA and sGAG assays across all experimental groups based on your dataset. For the DNA assay we used 5uL of a 500ul sample within each well to conduct the assay and the following standards were used:

For the GAG assay we used the following standards:

3) Statistical Analysis: Select an appropriate statistical test based on your dataset type. Perform the statistical analysis using GraphPad, include the results in your report, and provide a rationale for choosing that specific test.

4) Data Presentation and Discussion: Present your results graphically using GraphPad, including individual data points, error bars denoting standard deviation, and statistical annotations as applicable. Discuss your findings in the context of the bone/cartilage regeneration study, addressing how they support or challenge the hypotheses.

5) Lab Report Structure: Your lab report should include the following sections:

• • Introduction: Brief overview of the study’s aim and objectives, including a description of the experimental groups.
  • Materials and Methods: Explanation of biochemical assay procedures, data interpolation methods, and statistical tests applied.
  • Results: Presentation of interpolated data, standard curves, and graphs with statistical analysis.
  • Discussion: Interpretation of results, justification for statistical tests used, and relevance of findings to bone/cartilage regeneration.

6) Important Notes:

• • Half the class will focus on bone regeneration, while the other half will assess cartilage regeneration—ensure you follow the correct experimental conditions based on your assigned dataset.
  • Submit your final report by the due date; late submissions may incur grade penalties.
  • This assignment is designed to provide practical experience in analyzing experimental data and drawing conclusions based on statistical results.

Appendix:

1. Calcium SOP:

Calcium Standard Solutions (100 and 20µg/ml) (For one 96 well Plate):

NOTE: This Calcium solution comes as a stock with a concentration of 100 µg/ml (or 10mg/dL)

Under sterile conditions prepare a 100µg/ml and a 20µg/ml of Calcium standard solution as follows:

100µg/ml (or 100ng/µl)

• Take 100µl of 100 µg/ml Calcium stock solution and place in a 0.5 ml micro tube.
• Label tube.

20µg/ml (or 20ng/µl)

• Take 50µl of 100 µg/ml Calcium stock solution and place in a 0.5 ml micro tube.
• Add 200µl of 1M HCL (1 in 5 dilution). Final volume of 250µl.
• Label tube, vortex and spin in benchtop centrifuge.

Calcium Standards:

• Vortex and briefly spin Calcium standards (100 and 20µg/ml) in benchtop centrifuge.
• Prepare standards in labelled 0.5ml micro tubes as follows:

Working solution:

• Prepare enough working solution (140µL per well x96wells ~14ml per plate)
• Add 12ml Reagent 1 and 4.8ml Reagent2 (enough for one 96 well plate). Ratio is 5:2 of Reagent 1:Reagent2.

Note on sample dilution:

It may be necessary to dilute samples by a factor of up to 1000. Therefore, one should test several representative samples vs. STD7 by adding 140µL working dye solution to 10µL of both (see procedure below) and visually observing colour change. If the samples are darker than STD7, then dilute further.

Assay Procedure:

1. Each plate can accommodate 1 blank, 7 standards and 24 samples in triplicate
2. Place 10µl of each standard and sample into individual wells in triplicate in a clear round bottomed 96 well plate.
3. Add 140 µl of working dye solution using multichannel pipette (mix well).
4. Cover plate with tin foil and allow to incubate for 10mins at room temperature
5. Read in plate reader at 570 (560-580) nm absorbance using protocol on level 9.
6. Generate standard curve and taking into account sample dilution determine Calcium content for each sample.

2. ALP SOP:

Stop Solution:  3M NaOH in ddH₂O. For 50mL add 6g of MW 40 NaOH

1mg/mL pNPP (2.7mM) Solution:  Mix 1 tablet of p-Nitrophenyl phosphate (pNPP) and 1 tablet of TRIS buffer in 5mL of ddH₂O. This is enough for one plate (≈100 wells). OR. Mix 5mg of pNPP in 5mL of 1X Diethanolamine Substrate Buffer.

ALP Enzyme Solution:                   Add 4.3µL of ALP enzyme (1kU stock) to 995.7µL of ddH₂O. This gives a 4.3U/mL solution.

Keep samples, Enzyme and pNPP solution on ice during the assay.

Procedure:

1. Prepare 1X Diethanolamine Substrate Buffer by taking 1mL of 5X Diethanolamine Substrate Buffer into 4mL ddH2O.

2. Prepare the stop solution, 5mM pNPP solution and ALP enzyme solution.

3. Dilute 296µL of the 5mM pNPP solution with 504µL ddH₂O to generate 1mM pNPP standard. This solution is used to generate 0, 4, 8 12, 16, 20, 40 & 80nM/well pNPP standards. Prepare the pNPP standards in 1mL tubes as follows: *

4. Add 120μL of the standards to the appropriate wells.

5. Add 40μL of the samples to the appropriate wells. Bring the total volume up to 80 mL with ddH₂**

6. Add 50 μL of pNPP solution to each well that will contain the test samples.

7. Add 10mL of ALP enzyme solution to each well containing the pNPP standard and mix well. Incubate for 60 min at 25°C, protect from light.

8. Stop all reactions by adding 20mL of Stop solution into each standard sample reaction. Gently shake plate.

9. Measure O.D. at 405nm in a microplate reader.

10. ALP activity (U/mL) = A/V/T

Where A is amount of pNP generated by samples (in µM).
V is volume of sample added in the assay well (in mL).
T is reaction time (in minutes).

Suggested Plate-Layout*This protocol assumes samples are from media, if using cell lysate the standards should be diluted in the appropriate lysis buffer instead of media (H₂O if using the freeze-thaw method).

**More or less sample may need to be added in order for your readings to be within the standard curve, use a trial run to optimize the assay before running all your samples . The amount of media added to the standards will also need to be changed if the amount of sample per a well is changed (the calculations for creating the standards in step 2 are based on creating enough of each standard for 4 wells and using a sample volume of 40µL).

3. DNA SOP

1x TE Buffer (For one 96 well Plate):

Component B (Kept in press). Under sterile conditions, prepare as follows:

• 25ml of 20x TE stock buffer
• 75ml ultra pure H₂O

Working Reagent (For one 96 well Plate):

Component A (Kept in fridge). Prepare in minimal light as follows:

Note: Prepare in a 50ml falcon tube as solution will adsorb onto glass

• 22ml 1x TE buffer
• Remove 55µl of TE buffer (Keep extra 3ml for standards)
• Add 55µL PicoGreen stock solution (1 in 400 dilution)
• Label and wrap falcon tube in tinfoil
• Solution is light sensitive and should be used within a few hours of preparation

DNA Solutions (40 and 20µg/ml) (For one 96 well Plate):

NOTE: This DNA solution comes as a stock with a concentration of 100 µg/ml

Under sterile conditions prepare a 40µg/ml and a 2µg/ml DNA solution as follows

40µg/ml

1) Take 40µl of 100 µg/ml DNA stock solution and place in a 0.5 ml micro tube

2) Add 60µl of 1x TE (1 in 2.5 dilution)

3) Label tube, vortex and spin in benchtop centrifuge

2µg/ml

• Take 2µl of 40 µg/ml DNA stock solution and place in a fresh 0.5 ml micro tube
• Add 38µl of 1x TE (1 in 20 dilution). Final volume of 40µl
• Label tube, vortex and spin in benchtop centrifuge

DNA Standards:

• Vortex and briefly spin DNA (40 and 2µg/ml) in benchtop centrifuge. Prepare standards in labelled 0.5ml micro tubes as follows:

• Vortex standards and spin briefly in benchtop centrifuge

Assay Procedure:

• Each plate can accommodate 1 blank, 7 standards and 24 samples in triplicate
• Place 10µl of each standard and sample into individual wells in triplicate in a black flat bottomed 96 well plate Add 190 µl of working dye solution using multichannel pipette (mix well)
• Cover plate with tin foil and allow to incubate for 5mins in the dark at room temperature
• Read in plate reader at 480 nm excitation and 520 nm emmision
• Using standard curve and taking into account dilution extrapolate DNA content for each sample

Disposal

When finished the assay, pipette out all remaining solutions from the wells into a reservoir/trough and dispose of imediately in the non-chlorinated waste stream. Dispose of any contaminated plates, reservoirs, tips etc. immediately.

96 Well Plate Layout:

4. sGAG SOP

Prepare Standard as described below.

Procedure:

• Remove the desired volume from each papain digested sample and transfer to freshly labelled screw cap tubes.
• Final volumes must be the same. If necessary use PBE to make up to 100 or 200µl
• Add 1ml of DMMB (1, 9 dimethylmethylene blue dye) to each microtube
• Vortex and insert into the rotating wheel for 30 mins at 10r.p.m.
• Centrifuge microtubes at 15,000 X G for 10 minutes. Any GAG present in the sample will have bound to the dye and forced to the end of the tube to form a pellet which is purple in color.
• Remove microtubes and invert over some tissue paper and remove the excess dye on using a clean cotton bud.
• Add 1 ml of the dye dissociation agent to each microtube.
• Vortex or insert into the rotating wheel until the pellet has broken up.
• Setup the plate-reader and set the wavelength to 656 nm.
• Add 200µl each sample and standard (in triplicate) to clear round bottomed 96 well plate.
• Samples should not be left longer than 2 hours before taking measurements.

96 Well Plate Layout: