BME 201 Final Report: Bioreactor to

Test Mechanical Stability of

Synthetic Bone Graft

Clients: Dr. Amit Nimunkar and Dr. John Puccinelli University of Wisconsin ­ Madison

Department of Biomedical Engineering

Team Members: Rebecca Brodziski, Jack McGinnity, Ben Vander Loop,

Michael Weiser, and Brian Yasosky

BME 201: Lab 301, Team 1

4 May 2015

Table of Contents 1. Abstract 2. Introduction (Add block diagram and reference throughout)

a. Problem statement b. Background c. Project Motivation d. Hypothesis for the experiments to be performed

3. Design a. Temperature Sensor Circuit

i. Electronics Background ii. Initial Design iii. Final Design

b. Carrier Design i. Design One ii. Design Two iii. Design Three iv. Design Matrix

­ Design One Critique ­ Design Two Critique ­ Design Three Critique

v. Final Design c. Software

i. Arduino ii. LabVIEW

d. Biomaterials i. PMMA ii. Calcium Phosphate iii. Calcium Sulfate iv. Natural Material v. Biomaterial Matrix

­ PMMA Critique ­ Calcium Phosphate Critique ­ Calcium Sulfate Critique ­ Natural Material Critique

vi. Final Biomaterial 4. Methods/Testing

a. Fabrication of the carrier/holder

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b. Finite Element Analysis c. Temperature Calibration d. LabVIEW e. Biomaterial fabrication f. MTS/MATLAB

5. Results a. Data from Experiments (Tables) b. Statistics (T­test, graphs, average standard deviation) c. Young’s Modulus

6. Discussion/Conclusion a. Discussion of the results and the hypothesis for the experiments b. Cost/safety c. Ethical Consideration d. Sources of error

7. Future Work/Design Improvements 8. References 9. Acknowledgements 10. Appendix

a. Product Design Specification b. Extra Data c. Code d. Circuits Schematics (Diagram)

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1. Abstract Bone graft implants, which can be used to stimulate new bone growth in patients with injuries or bone defects, are currently the second most transplanted material worldwide. With close to 2.2 million procedures a year, bone grafting generates sales of approximately $2.5 billion [7]. Over half of these procedures are done with samples obtained from the patient (autograft) or from another human donor or cadaver (allograft) [8]. There are a number of utile synthetic biomaterials that can be used for bone grafting, and have the ability to avoid problems such as disease transmission, infection, or lack of bone growth. Because of this they are very valuable, and it is worth while to test their viability as replacements for bone. The team was tasked with creating such a material that would replicate the properties of natural bone. However under bodily conditions the mechanical and structural properties are subject to degradation. This required the fabrication of a system which can replicate the environment that the grafts would experience in vivo. In order to do so, the client asked for a bioreactor that would be able to hold the bone samples inside for up to two weeks at body temperature, in a medium of phosphate buffered saline (PBS). A sample holder was fabricated to hold the samples, and a microprocessor was used to acquire temperature data and maintain the temperature within the bioreactor. After one day and one week, the samples were removed from the bioreactor for MTS compression testing. The results of the MTS testing showed that there was no significant difference in the average Young’s modulus among the three timepoints for which the bone grafts were tested. Therefore, the samples did not significantly deteriorate over the time of testing. However, the Young’s modulus for the bone graft samples was significantly different from that of natural bone, which implied that the samples fabricated were not an adequate substitute for natural bone from a mechanistic view. 2. Introduction

a. Problem Statement Professors Amit Nimunkur and John Puccinelli have provided the task of creating a

bioreactor that could simulate bodily conditions to test a synthetic bone graft. The bone graft samples must fill a hole of 14 mm diameter and 20 mm in height inside a human femur. Additionally, they must maintain mechanical properties after a minimum of two weeks in the vessel. Mechanical testing was to be performed at three time intervals to determine if deterioration was occurring under bodily conditions. The bioreactor was to fit inside a corningware jar with the dimensions shown below in Figure 1. The jar will be filled with a Phosphate Buffered Saline (PBS) solution and maintain a constant temperature of 37.5°C (± 5°C) to mimic body conditions. The holder inside the bioreactor must be able to fit six samples. This design was to be

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accomplished within a fourteen­week period and under a $150 budget using the jar, an Arduino microcontroller, LabView, and basic circuit components. Further design specifications can be found in section 10.a of the appendix.

Figure 1. Corningware jar used to hold the bioreactor. All dimensions are in mm. b. Background

A tissue engineering bioreactor is a device in which biological or biochemical processes develop under a closely monitored and tightly controlled environment [1]. They are used in tissue engineering for the fact that the environmental stimulation provided by the reactors stimulates the cells and encourages them to produce extracellular matrix at an increased rate. Although bioreactors may range from large industry applications to small scale lab work, the specific function of the reactor remains the same. It must be able to maintain a physiological environment close to a temperature of 37, a pH of 7.4, as well as monitor oxygen levels and nutrient concentration [1]. However, the bioreactor created in this project will only regulate temperature and pH, as well as be sealed from the external environment.

Human bones undergo longitudinal and radial growth, modeling and remodeling throughout life [2]. They also can change their shape to accommodate for stresses that are placed on them. The presence of mineral salts in the osteoid matrix, a crystalline complex of calcium and phosphate, provides bones with their rigidity and hardness [3]. Due to the fact that new bone formation can take up to 6 months to complete, synthetic bone use has become a helpful tool to aid in bone regrowth. Synthetic bone samples selected for implantation within a patient must have a degradation rate that is similar to the cellular rate of bone formation to allow for proper stability and strength [4]. These samples must also allow the body to naturally absorb them as to not cause complications.

3. Design a. Temperature Sensor Circuit

The team was required to design a circuit to control a heating element to maintain a constant temperature within the bioreactor. This incorporated the use of a thermistor, which is a resistor that changes its value for resistance with temperature. This circuit was required to measure the voltage change that results as the resistance changes in the thermistor based on the temperature inside the bioreactor. The circuit was powered by an Arduino Uno© microprocessor, which also takes a voltage output from the circuit and converts the value to a temperature (see section (c) within Design). In designing this circuit, it was assumed that the

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resistance of the thermistor was 10kΩ, with a variance of ±2kΩ. The thermistor was carefully calibrated using water baths of different temperatures and recording the voltage as read by the Arduino software. The circuit was also designed so that the output voltage from the circuit would fall somewhere within the range from 0­5V and then this voltage value could be processed using the code written. [locate in appendix?]

i. Electronics Background Basic circuit components such as thermistors, resistors, and a breadboard were used.

A thermistor was used because it has varying resistance with temperature which allowed it to act as a temperature sensor. In order for the thermistor to act as an accurate temperature sensor, it must be calibrated based on voltage readings from a special circuit. This procedure is outlined in section 4c Thermistor Calibration. Another resistor was used to complete the voltage divider part of the circuit. An additional resistor is needed to prevent the delicate led from frying due to the 5.0 V it receives from the Arduino board. All of the previously described electronics principles were implemented into this project.

ii. Initial Design The preliminary design created by the team incorporated a voltage divider,

followed by a low­pass filter, whose Vout was then sent to the microprocessor. The current from the DC voltage source of 12 V was designed to first pass through the thermistor.

Assuming that the temperature of the human body is 37°C, the resistance of the thermistor at that temperature was calculated to be 6,047.38Ω. This was performed by first determining the proportional resistance decrease from 35°C to 40°C using the thermistor datasheet [citation].

dif ference 529.7Ω 323.9Ω 205.8Ω = 6 − 5 = 1 Since 37­35=2, and ⅖ = 0.4, the proportional resistance was subtracted from the resistance value for 35°C. esistance 6529.7Ω (1205.8Ω 0.4) 047.38ΩR = − × = 6

In order to leave a 1V buffer zone, 4V was chosen as the output voltage. The input voltage was arbitrarily chosen to be 12V as the input arbitrarily, as it was unclear what the source of power was for the circuit at the time at which this circuit was designed. To determine the resistance of the second resistor in the voltage divider, R2, the following equation was used:

4=12V(R2/(R2+6047.38Ω)) The calculated R2 was determined to be 3,023.69 . This value was roundedΩ

down to 3000 Ω and inserted back into the previous equation to calculate a more specific input voltage by computing the following:

4=Vin(3000Ω/(3000Ω+6047.38Ω) The newly calculated value for Vin was determined to be 12.0632V.

To construct the low­pass filter, the following formula was used:

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2π(60hz)=1/((1*10^­6F)(R)) The capacitor value of one µF was arbitrarily chosen, which gave a resistance R of 2.65k ; therefore, a 2.7kΩ resistor was chosen for use in the circuit.Ω

The voltage from the low­pass filter was designed to be sent to the microprocessor, which reads the voltage. If it is greater than 4.0V, it will trigger the heating element to turn off. If the voltage is less than 4.0V, the heating element will turn on. A diagram of this preliminary design is outlined in Figure X.

iii. Final Design

The final design pictured above was decided on because it accomplished the task in the most efficient manner possible. The low­pass filter that was included in the initial design was found to be unnecessary after reviewing feedback on the electronics report. At one point adding an operational amplifier was considered but this was not needed because circuit worked correctly and was within the temperature tolerance range without one. Careful calibration and testing was done to verify this. The bioreactor had a measured temperature of 33.2°C which is within the tolerance range of 37°C +/­5°C.

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b. Carrier Design Another facet of this project was to create a biomaterial holder that was capable of

holding up to six synthetic bone samples at a time, and make sure that the samples could not become dislodged. The primary considerations were efficacy, cost, ease of manufacture and safety. The holder also needed to be non­reactive with the samples, as well the PBS medium for a period of two weeks.

i. Design One Design one was the simplest of the three holder designs, and was intended to meet the

design requirements while being very simple and inexpensive to fabricate. It consists of 6 wells arranged in a circle that would hold the samples towards the bottom of the reactor. The wells would have a radius of 15 mm, which is just wide enough to accommodate the samples. All of the samples would be held at the same depth, which helps control the temperatures they are exposed to. However, the fact that the holder would rest on the bottom of the

corningware jar makes removing the samples for testing difficult. Another consideration for this design was that the bottom of the samples would not be in contact with the solution as much as the tops and sides would. ii. Design Two

This design was a bit more complex, and had a base similar to a wine glass. The idea was to support the holder and elevate the samples so they were centrally located in the reactor. Again, the samples

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The major improvement on this design compared to Design One was that this design

incorporated elevating the wells for the bone samples so the disc wasn’t sitting on the bottom of the bioreactor. This design includes a thin, disc­like base with a rod protruding from the center of it; this part was designed to sit at the bottom of the bioreactor and provide a base for the sample holder. The top part was designed as a thicker disc with 6 wells to hold the bone samples. This disc had a hollow rod extending from the bottom face of the disc which mated with the rod from the bottom part of the holder, telescoping so that the top part can slide onto the bottom part of the holder. The top part can then freely rotate as it sits on the rod from the bottom part.

iii. Design Three

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This design incorporates elements from Design Two; it has a thin disc at the bottom to

provide a base for the sample holder, and has a thicker disc at the middle of its height with six wells that hold the bone graft samples. Additionally, at the very top of the part is a thinner disc that has four large holes, which function as a sort of handle by which to grab the sample holder when placing it in and out of the bioreactor.

iv. Design Matrix

Design 1 Design 2 Design 3

Accuracy (25) (5/5) 25 (5/5) 25 (5/5) 25

Machinability (20) (5/5) 20 (3/5) 12 (3/5) 12

Durability (15) (5/5) 15 (5/5) 15 (5/5) 15

Convenience (20) (1/5) 4 (4/5) 16 (4/5) 16

Cost (10) (5/5) 10 (4/5) 8 (4/5) 8

Safety (10) (5/5) 10 (5/5) 10 (5/5) 10

Total (100) 84 86 86

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­ Design One Critique

The major flaw in this design is ease of use when placing the sample holder into and out of the bioreactor. There is no sort of handle available for use when moving the holder. Additionally, this holder would sit at the very bottom of the bioreactor, which would make it more difficult to retrieve from the bioreactor to remove samples.

­ Design Two Critique This design places the bone graft samples higher in the bioreactor, but

still lacks a sufficient handle with which to retrieve the sample holder from the bioreactor. Additionally, the way this design mates would make it difficult to align within the bioreactor, since only the top part would be able to be retrieved from the bioreactor and the bottom part would remain within the bioreactor.

­ Design Three Critique This design resolves the issue of lack of a handle, by providing finger

holes at the top of the holder. However, the fabrication of this holder would be difficult. As is designed, this sample holder consists of one piece, not multiple. It would be incredibly difficult to drill the holes to hold the bone samples as well as the holes for the handle.

v. Final Design The ultimate design that was chosen represents the iterative nature of the design process. It was decided that it would be convenient to have the samples being tested

for different durations housed separately in the bioreactor. That led to the creation of a fourth design, pictured left. The holder would have two separate “pucks” which each contained 3 wells to hold the samples. They would be held together by a third component, a rod of variable thickness that was threaded on one end. This would allow for easy removal of the week one samples, without disturbing the ones held in the bottom puck. However, during fabrication the rod component was subjected to a torsional force when being threaded. The die dug into the HDPE and stuck, causing a shear failure on the region that was supposed to be threaded (figure #,#). A ¼ in diameter stainless steel rod with 20 threads per inch was purchased. The rod was then cut to 98 mm to replace the HDPE rod. This was successful, and the phosphate buffered

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saline did not react with the stainless steel. The final assembly can be seen in (figure #)

c. Software

i. Arduino The basic purpose of the Arduino microcontroller and code is to read voltage

from the thermistor circuit [SEE FIGURE X] and convert this voltage into a temperature in °C. This temperature value is then passed through a conditional statement that checks if the current temperature is less than, greater than, or equal to 37°C. If the temperature is greater than or equal to 37°C the arduino board will signal to the heating element to stay off because the bioreactor is warm enough. If the temperature value is read as anything less than 37°C, the Arduino will signal the led and the beefcake relay to turn on. This temperature is checked 4 times per second and then adjusted according to the previously described. Finally, there is an outermost

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loop where the Arduino code checks the rocker switch position and if it is on the previously described internal condition will be checked but if it is off nothing will happen and the system will be inactive. The final Arduino code can be found in appendix 10c. ii. LabVIEW

The LabVIEW software was used to take the temperature output from the Arduino and produce a graph output from that data. It was also used to save this data to a spreadsheet for recording the change in temperature over time.

d. Biomaterials

i. PMMA PMMA has been found to be one of the most widely used polymers in tissue

grafting because of its moderate properties, easy handling and processing, and low cost. The use of PMMA increases the load­bearing and fixation capacity of the reconstruction site by transferring the weight of the body into this bone cement. However, PMMA is also very dense and because of this, does not allow for much bone in­growth. It is also non­degradable making it a permanent implant which increases the risk of an immune response. [citation]

ii. Calcium Sulfate Calcium sulfate is another widely known material in tissue grafting. Although

it too is known to be quite dense, it still provides a compact, crystalline structure to which both osteoblasts and osteoclasts have been shown to attach. It also lends mechanical stability and tensile strength to the bone graft. Unlike PMMA, bone grafts of calcium sulfate are biodegradable and have been shown in vivo to dissolve within about 8 weeks, providing a temporary scaffold onto which new bone can grow and making it one of the fastest degrading osteoconductive materials. [citation].

ii. Calcium Phosphate

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Calcium Phosphate also provides a crystalline structure conducive to supporting growth of new bone, and has been shown to be resorbable in vivo. It has a slower degradation rate compared to Calcium Sulfate, but still has the ability to provide adequate mechanical support. Like Calcium Sulfate, Calcium Phosphate also displays excellent biocompatibility and is able to promote osteoconduction and osseointegration.This compound has also been been shown to display good bonding to natural bone, with direct contact to bone being observed. [citation]

iv. Natural Material There are several types of natural biomaterials that have been utilized in bone

grafting, including autografts, allografts and xenografts. Autografted material comes from a donor site on the patient, and because of this has the lowest incidence of immune rejection. However, this creates a trauma to the harvesting site of the patient’s body, which can cause discomfort and extended hospital stay time. This issue can be circumvented by taking the bone tissue from another person. The typical source for allografted bone material is from a cadaver, which is non­living tissue. This means that it can provide a scaffold for healthy cells to culture and regrow, but the grafted material lacks the proteins and cells necessary for sufficient bone growth. Additionally, since the bone is not from the patient who is receiving it, there is a risk for disease transmission. Another natural type of graft used for bone replacement are xenografts. This involves the use of material from other species, typically bovine, porcine, or coralline. The coralline replacement is mostly calcium carbonate, which can be converted to hydroxyapatite (Springer). If left in calcium carbonate form, the material will be more easily re­absorbed by the natural bone material, but if converted the graft will not be re­absorbable. This is very similar to the calcium carbonate that was used to fabricate the synthetic bone, and the properties should be comparable.

v. Biomaterial Design Matrix

Material: Calcium Phosphate (β­TCP)

Hydroxyapatite and (β­TCP) 60:40 Blend

Calcium Sulfate and Calcium Carbonate (3:1 final ratio)

Strength (25) (5/5) 25 (3/5) 15 (4/5) 20

Lifetime (25) (5/5) 25 (5/5) 25 (4/5) 20

Resorption Rate (20) (1/5) 4 (2/5) 8 (5/5) 20

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Safety (20) (4/5) 16 (4/5) 16 (4/5) 16

Cost (10) (3/5) 6 (2/5) 4 (5/5) 10

Total (100) 76 68 86

­ PMMA Critique

The biggest downside to PMMA was that it is not biodegradable and so there is a much greater chance of a harmful immune response.

­ Calcium Phosphate Critique Calcium phosphate was not chosen because it has a slow resorption rate of

approximately three years. ­ Calcium Sulfate and Calcium Carbonate Critique

Calcium Sulfate does not have a very long lifetime. However, since the samples only need to be in the bioreactor for two weeks, this was not seen as a major problem. When combined with Calcium Carbonate, the structural strength and longevity is also increased to attempt to solve the previous problem.

­ Natural Material Critique The drawback to natural materials such as autografts and allografts is that they

are expensive and require surgical extraction. These drawbacks mean that natural materials are far out of the reach of this class for obvious reasons. vi. Final Biomaterial Calcium Sulfate blended with Calcium Carbonate was chosen since it had the highest

score on the design matrix. The only drawback was its short lifetime but this did not matter since the time spent in the bioreactor was only to be two weeks long. If the circumstances were different, this decision may require some reevaluating.

4. Methods/Testing

a. Fabrication of the carrier/holder To fabricate the bone graft holder, one half­foot of 2.5­inch diameter rod of

high­density polyethylene and 9 cm of ⅜­inch diameter threaded rod of stainless steel were used. The plastic rod was turned down to a diameter of 60 mm using a lathe, and then was cut into discs of a minimum height of 30 mm using a bandsaw. Each disc was faced on the lathe on both sides to achieve an even surface, and to decrease the height of the discs to 25 mm. A through hole was drilled through the center of one disc using a number 7 drill bit(?).

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On the other disc, a hole was drilled in the center using the same drill bit size, but only to a depth of 10 mm. Each hole was tapped using a ¼­20 tap.

Three holes were drilled onto the top face of each disc, in order to hold the bone graft samples within the bioreactor. A 41/64 inch drill bit was used, and holes were drilled to a depth of 5 mm. The three holes were all placed around the center of the disc, so that they were all equidistant from the center hole. The threaded rod was then twisted into the bottom disc, the disc without the through center hole. The disc with the through hole was then twisted over the top of the threaded rod so that there was a 25 mm space between the two discs. b. Finite Element Analysis ??? ANYONE KNOW WHAT THIS SHOULD CONTAIN ??? c. Temperature Calibration

To calibrate the thermistor, the circuit outlined in the final design for the temperature sensor circuit (see section (a.iii.) within Design) was constructed and wired to the microprocessor. The preliminary code for acquiring voltage data from the circuit (see section (c.i) within Design) was uploaded to the microprocessor. The thermistor was submerged in water baths of known temperatures, and the voltage reading at each temperature was recorded. This data was plotted to form the calibration curve shown in Figure X. Since the R2 value for the curve was determined to be 0.9981, it was assumed that the plot was linear, and that the linear regression line could be used in converting voltage data to temperature data. The equation for this conversion was determined to be:

T = ­0.1073(ADC output) + 110.77 This equation was then used in the final microprocessor code in order to send temperature data to the LabVIEW software.

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Figure X. Plot of the voltage output from the thermistor circuit against the temperature to which the thermistor was exposed. The thermistor was submerged in water baths at temperatures ranging from 20°C to 60°C, at intervals of 5°C. d. LabVIEW

LabVIEW software was used to record the temperature data output by the microcontroller. A VISA Configure Serial Port VI was used with VISA Read within a while loop to input the serial communication of temperature data from the microprocessor and output it to a graph indicator. The Build Waveform function was used to add a time scale to the graph output by the VISA Read. Additionally, Build Array was used to create an array from the data points output by the VISA Read. Since the sampling rate of the microprocessor was 4 Hz, the time interval between data points on the graph displayed by LabVIEW was ¼ second. Finally, the data that was output to the graph indicator was also output to be saved to a spreadsheet. The completed VI can be seen in Figure X. e. Biomaterial fabrication

A ratio of 75:25 of CaSO4 to CaCO3 was desired. However, in addition to primarily calcium sulfate, the Plaster of Paris(POP) mixture that was used contained up to 10% of calcium carbonate. For this reason, the ratio of POP to calcium carbonate was converted to 90:10. To achieve this ratio, 18 g of Plaster of Paris powder was used for every 2 g of CaCO3 powder to form a 20 g mixture of powder. This powder was mixed in a plastic cup, and 40 mL of deionized water was added to create a 2:1 ratio of liquid to powder. This procedure was repeated three times. The mixture was then transferred to wells 14 mm in diameter and 20 mm deep, and was packed and smoothed out to form the bone graft sample. Bone samples were allowed to harden overnight on the bench. The finished samples ranged in quality with a few having uneven edges and air pockets. Since a total of twelve samples were made and only nine were needed, the 3 poorest­quality samples were discarded.

Mixture 1 Mixture 2 Mixture 3 Totals:

Plaster of Paris 18.00 g 18.01 g 18.01 g 54.02 g

Calcium Carbonate

2.00 g 2.00 g 2.01 g 6.01 g

f. MTS/MATLAB Performing the MTS compression analysis is done by first measuring the diameter of

each sample. Each sample was placed onto the MTS and the crosshead was lowered so that it was just above the sample. The load reading was zeroed and the test was started. The graph of Stress vs. Strain and the sample itself were watched to make observations about how the sample responded. When the strain value stopped decreasing, the test was stopped and

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the data was saved to analyze with MATLAB. Using MATLAB the raw text file was loaded and the headings were ermoved so that only a 3 column array remained. Each column was then extracted for force and displacement data. This data was then fed into equations for Stress and strain. The cross­sectional area was calculated for each sample and added in its own line of MATLAB code so that the stress could be calculated. A plot of Stress vs. Strain was created in MATLAB for each of the three data sets.

5. Results

a. Data from Experiments (Tables) After the samples were made, an MTS was used to plot stress vs strain. This data was

analyzed with a MATLAB script to obtain the young’s modulus. This was done for three samples that were never in the bioreactor, three samples that were in the bioreactor for one day, and three samples that were in the bioreactor for one week. The full data is shown in the appendix and the averages with standard error are shown in Figure x below.

Figure x. Average Young’s modulus for three test times. Initial samples were never in the Bioreactor, T1 samples were in for one week, and T2 samples were in for two weeks.

In order to determine if any significant difference in Young’s Modulus was present, an ANOVA was performed with a Fisher’s LSD test between Initial and T1, Initial and T2, and T1 and T2. All p­values were >0.05 indicating that no there is no significant difference in Young’s Modulus between any test times.

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6. Discussion/Conclusion

a. Discussion of the results and the hypothesis for the experiments Since no significant difference in young’s modulus was observed between the test

times, the data indicates that the samples did not decay in the bioreactor. However, the quality of the samples varied greatly with some containing numerous air pockets. The three samples with the most air pockets were chosen for initial testing and the six best were saved for the bioreactor. Since air the air pockets would decrease mechanical stability, this could explain why the average young’s modulus increased from the initial test to T1.

The Young’s Modulus of compact bone is about 11­21 GPa (University of Cambridge). After one week in bodily conditions, the samples’ Modulus was significantly less at an average of 79.8 MPa. This indicates that the Calcium Sulfate samples would be much weaker than the rest of the bone if inserted. b. Cost/safety

The total cost of this project was $109.8 and the budget breakdwon is shown in Table x in the appendix. All materials were supplied except for materials used in fabrication. Overall, the goal of staying under the $150 budget for designing, building, and testing the synthetic bone graft material was met.

Several measures were taken to ensure safety throughout all aspects of the design process. Whenever samples were placed in the bioreactor or removed, a biosafety cabinet was used to ensure all samples remained sterile. Moreover, all circuit components were contained inside a sealed box to prevent any electrical issues if the PBS solution were to leak. Also, an LED light was included in the circuit to alert anyone nearby that the heating element was on. c. Ethical Consideration

While designing the bioreactor, there were many considerations made to assure that the final product would be safe for the client and future users. The product includes a power switch that is more convenient than pulling the plug out of the wall each time the user wants to turn off the device. Additionally, a base heating element was chosen as opposed to the wrap around heating element because it is safer in regards to accidental burns. The internal environment of the bioreactor was designed to simulate that of a human body in terms of pH to ensure the samples wouldn’t react in harmful ways to the body. To further ensure safety, careful consideration went into picking the materials for this project. Some of the requirements were biodegradability, source of the provider, cost, and quality/durability. In the future one could consider making the jar out of a heat resistant plastic to prevent shattering in the event that it is dropped.

7. Future Work/Design Improvements

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One of the biggest challenges was maintaining consistent quality among the bone graft samples. Packing the Calcium Sulfate paste down as it is being put into each well will eliminate air bubbles. Also, carefully smoothing out the top of the paste before it solidifies will ensure that all samples come out smooth and even. The circuit could be improved by soldering all components together and purchasing pre­cut, thicker wire. This would tighten up the circuit and help reduce the chance of short­circuiting. In the sample holder, the holes drilled for the samples were not flat as a drill­bit with a lead was used. Using a bit with a flat head would ensure a flat surface for the samples to rest on. Moreover, with extra time the samples could be tested in the bioreactor for a full two weeks.

8. References

1. S. Partap, N. A. Plunkett and F. J. O’Brien (2010). Bioreactors in Tissue Engineering, Tissue Engineering, Daniel Eberli (Ed.), ISBN: 978­953­307­079­7, InTech, DOI: 10.5772/8579. Available from: <http://www.intechopen.com/books/tissue­engineering/bioreactors­in­tissue­engineering>

2. "Introduction to Bone Biology: All About Our Bones." International Osteoporosis Foundation. 2015. Web. <http://www.iofbonehealth.org/introduction­bone­biology­all­about­our­bones>.

3. Clarke, Bart. "Normal Bone Anatomy and Physiology." Clinical Journal of the American Society of Nephrology : CJASN. American Society of Nephrology, 3 Nov. 2008. Web. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152283/>.

4. Fröhlich, Mirjam, Warren L. Grayson, Leo Q. Wan, Darja Marolt, Matej Drobnic, and Gordana Vunjak­Novakovic. "Tissue Engineered Bone Grafts: Biological Requirements, Tissue Culture and Clinical Relevance." Current Stem Cell Research & Therapy. U.S. National Library of Medicine, 5 Nov. 2009. Web. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773298/>.

5. 'Bone Grafts and Bone Graft Substitutes in Periodontal Therapy' p.92 Chapter 2.3.3 'coralline calcium carbonate', Dumitrescu et al, 2011, Springer ,ISBN 978­3­642­18224­2

6. "Mechanical Properties of Bone." ­ TLP Library Structure of Bone and Implant Materials. University of Cambridge, Web. 03 May 2015.

7. Lewandrowski KU, Gresser JD, Wise DL, Trantol DJ. Bioresorbable bone graft substitutes of different osteoconductivities: a histologic evaluation of osteointegration of poly(propylene glycol­co­fumaric acid)­based cement implants in rats. Biomaterials 2000; 21:757­764.

8. A.A. Jahangir, MD; R.M. Nunley, MD; S. Mehta, MD; A. Sharan, MD; (2008) Bone­graft substitutes in orthopaedic surgery [Online] AAOS, http://www.aaos.org/news/aaosnow/jan08/reimbursement2.asp

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9. Acknowledgements Special thanks to Professors Amit Nimunkar, John Puccinelli, and Joseph Towels for their guidance through the design process. We greatly appreciate the help we received from the student shop personnel in order to properly fabricate the bioreactor components. Also, thanks to TA Matt Jensen and all of the SA’s who taught us the skills to make this project possible. 10. Appendix

a. Product Design Specification Function:

The bioreactor must maintain conditions similar to average biological conditions within the human body, in terms of temperature, pH, pressure, etc. These conditions must be maintained for a period of at least 2 weeks. A sample holder must fit within this bioreactor and must hold at least 6 bone graft samples at a time. An external computer must record temperature within the bioreactor, and a sensor and heating element must maintain a stable temperature. Artificial bone graft samples must maintain mechanical stability similar to natural bone in vivo for at least 2 weeks.

Client Requirements: 1) The entire project needs to cost less than or equal to $150.00. 2) The final product must be capable of maintaining a bone graft in physiological conditions

(temperature, pH, and pressure) for a minimum of 2 weeks. 3) The resulting bone graft must be similar in structural strength and density to natural bone. 4) The bioreactor must be large enough to hold at least 6 bone graft samples simultaneously. Physical and Operational Characteristics: Performance Requirements: The product must be able to create a synthetic bone graft that is 20mm deep with a 14 mm diameter. The bone graft must be able to survive in vivo for at least two weeks. It also needs to have similar rigidity and structural strength to that of natural bone. Safety: The bone graft and bioreactor cannot have any hazardous or radioactive materials. The device must also be sanitary so that the bone graft can be properly sterilized before it is potentially used on a patient.

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Accuracy and Reliability: The bone graft must have similar structural characteristics to natural bone. It will be tested in tension using an MTS Sintech Tensile testing machine to determine its tensile strength. The bone graft must be strong at the beginning of its life and after two weeks in the bioreactor. The bioreactor must accurately and consistently maintain a 98.6°F internal environment similar to physiological conditions. Life in Service: The bone graft must survive in the bioreactor for a minimum of two weeks. The client may desire that the bone graft last significantly longer in a potential patient. Shelf Life: The bioreactor must have a shelf life of at least 10 years, and all components should remain accurate within this time period. Operating Environment: The operating environment will be in the BME Design Studio, or some like facility in ECB. The bioreactor should be able to operate in any like conditions (such as in a laboratory, classroom, etc.) Environment within bioreactor must correspond to normal biological conditions within the body, such as temperature, pH, etc. Ergonomics: Bioreactor must be easy enough for use by knowledgeable individuals. Must not be too complex to maintain conditions within the bioreactor or to access the bone sample. Size: Bioreactor must be constructed using a 500 mL Corningware jar. Artificial bone graft must have 20mm depth and 14mm diameter. A sample container to hold at least 6 bone graft samples at once within the bioreactor must be large enough to hold the samples, yet small enough to fit within the jar. Power Source: The temperature sensor and heating element will be powered from the wall power supply. Weight: No weight is specified for the bioreactor, but it must be light enough to be moved and handled easily by one person. The artificial bone graft must be of a similar weight as natural bone of the same volume. Materials: The client has not specified a particular material to make the bone graft from, but will provide the team with options to choose from. The bone graft designed from any of the materials must maintain mechanical stability similar to native bone in vivo for at least two weeks. Aesthetics, Appearance, and Finish: The final product should be aesthetically pleasing in a way that it does not hinder functionality. Production Characteristics: Quantity: One bioreactor shall be constructed and at least 6 units of bone grafts. Target Product Cost: The targeted cost for this product is less than or equal to $150

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Miscellaneous: Standard and Specification: The device must comply with all the relevant FDA regulations and due to the fact that the device shall be entering the body, it must be cleared as a Class III device by the FDA. Patient­Related Concerns: The patient concerns would include reliability of device function, if they have any allergies to the product, sterilization, location of injury, severity of injury, and overall patient safety.

Competition: There are a few similar devices in the market currently including Medtronic’s

Infuse® Bone Graft/LT­Cage. These existing devices work by being surgically placed where

new bone is needed, and attracts the body’s own bone­building cells to build bone over time. Customer: A start up company is looking to design its own bone graft and would like the product to be affordable. b. Extra Data

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Item Amount Cost

Miscellaneous

heating pad 1 $24.0

jar 1 $9.2

breadboard 1 $4.5

rocker 1 $0.5

leds 1 $0.5

box 1 $8.4

power cord 1 $7.0

usb cables 1 $4.0

led grommets 1 $0.5

arduino 1 $25.0

Biomaterials

Plaster of Paris 54g $0.1

CaCO3 6g $0.1

24 well plate 1 $1.1

Construction materials

2.5 in HDPE rod 1 $14.0

1 ft stainless steel threaded rod

1 $1.1

3/8 in HDPE rod 1 $0.9

Circuit material

relay 1 $8.0

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thermistors 1 $1.1

Total Cost $109.8

Table X. Cost of all materials used in design, fabrication and testing. The total cost was successfully kept under the $150 budget.

c. Code // Bioreactor Arduino Code //Initialize our pins/variables float voltageReading = 0; int thermistor = A0; int heatLED = 7; //Tells if heating element is on int rockerSwitch = 2; int Beefcake = 4; //turns on/off heating element int SetTemp = 36; // Desired temp value void setup() pinMode(heatLED, OUTPUT); pinMode(Beefcake, OUTPUT); pinMode(rockerSwitch, INPUT); Serial.begin(9600); void loop() if (digitalRead(rockerSwitch) == 1) float raw = analogRead(thermistor); // Read the analog pin float temp = ­.1073 * raw + 110.77; //convert ADC to temperature Serial.println(temp); //send temp data to serial out if (temp < SetTemp) //check actual temp with set temp digitalWrite(Beefcake, HIGH); digitalWrite(heatLED, HIGH); else if (temp >= SetTemp) //turn off condition

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digitalWrite(Beefcake, LOW); digitalWrite(heatLED, LOW); delay(250);// Sets the sampling rate to be 4 Hz else if (digitalRead(rockerSwitch) == 0) // do nothing if rocker is off digitalWrite(heatLED, LOW); digitalWrite(Beefcake, LOW);

d. Circuits Schematics (Diagram)

%%Stress vs Strain Curve %First Sample ask = uigetfile('.txt','Select file'); MTS1 = load(ask); disp1 = MTS1(:,1); load1 = MTS1(:,2); time1 = MTS1(:,3); disp0_1 = 20; %height of sample % calculate values strain1 = (disp1)/disp0_1; r_squared1 = (15.5/2)^2; CSarea1 = pi*r_squared1; stress1 = load1/CSarea1;

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%Second Sample ask2 = uigetfile('.txt','Select file'); MTS2 = load(ask2); disp2 = MTS2(:,1); load2 = MTS2(:,2); time2 = MTS2(:,3); disp0_2 = 20; % calculate sample 2 values strain2 = (disp2)/disp0_2; r_squared2 = (15.5/2)^2; CSarea2 = pi*r_squared2; stress2 = load2/CSarea2; %Third Sample ask3 = uigetfile('.txt','Select file'); MTS3 = load(ask3); disp3 = MTS3(:,1); load3 = MTS3(:,2); time3 = MTS3(:,3); disp0_3 = 20; strain3 = (disp3)/disp0_3; r_squared3 = (15.5/2)^2; CSarea3 = pi*r_squared3; stress3 = load3/CSarea3; %Plot above data hold on plot(strain1,stress1,'­­r') plot(strain2,stress2,'­b') plot(strain3,stress3,'k') title('Plot of Stress vs Strain') xlabel('Strain') ylabel('Stress (MPa)') legend('Sample 1','Sample 2','Sample 3','Location','southeast') hold off

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%red j1 = ginput(1); j2 = ginput(1); E1 = (j2(2)­j1(2))/(j2(1)­j1(1)) %blue j3 = ginput(1); j4 = ginput(1); E2 = (j4(2)­j3(2))/(j4(1)­j3(1)) %black j5 = ginput(1); j6 = ginput(1); E3 = (j6(2)­j5(2))/(j6(1)­j5(1))

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