Final Report

Ultrasound Mediated Non-Union Fracture Repair System

Team 21

Nicholas Calistri, Aimon Iftikhar, Yvonne Nanakos and Kelly Stratton

TA: Sarah Brittain

Faculty Advisor: Dr. Krystyna Gielo-Perczak

Project 40

for

Dr. Yusuf Khan

University of Connecticut Health Center

Department of Orthopaedic Surgery

Department of Chemical, Materials & Biomolecular Engineering

Biomedical Engineering Program

MC3711, Room No: E7052

263 Farmington Avenue

Farmington, CT 06030

Phone: (860)-679-4097

Table of Contents:

Abstract...........................................................................................................................................3

1. Introduction.................................................................................................................................4

1.1 Background (Client and Disability)………......………..……………...……....….....4

1.2 Purpose of the Project…………………….......………………………………......…4

1.3 Previous Work Done by Others…………………………………………………......5

1.3.1 Products…………………..……………...…….……………………..…...6

1.3.2 Patent Search Results………………………………..………………...….6

1.4 Map for Rest of Report………………………………….………………………….7

2. Project Design.............................................................................................................................8

2.1 Introduction...................................................................................................................8

2.1.1 Alternative Design One: PLGA Scaffold……………………………….....8

2.1.2 Alternative Design Two: Hydroxyapatite Scaffold………………………..9

2.1.3 Alternative Design Three: Alginic Hydrogel Scaffold………………..….10

2.2 Optimal Design............................................................................................................13

2.2.1 Objective…...................................................................................................13

2.2.2 Controlled Stimulation ….............................................................................15

2.2.3 Material for Hydrogel …..............................................................................18

2.2.5 Application with Osteoblasts........................................................................18

2.2.6 Integration of all Components into Device...................................................19

2.2.7 SolidWorks Simulation.................................................................................20

3. Realistic Constraints…………………………………………………………………………..22

4. Safety Issues…………………………………………………………………………………..24

5. Impact of Engineering Solutions ……………………………………………………………..25

6. Life-Long Learning…………………………………………………………………………...26

7. Budget and Timeline………………………………………………………………………….27

7.1 Budget……………………………………………………………………………….27

7.2 Timeline……………………………………………………………………………..29

8. Team Members’ Contribution to the Project…………………………………………………31

9. Conclusion……………………………………………………………………………………33

10. References…………………………………………………………………………………...34

11. Acknowledgements………………………………………………………………………….35

12. Appendix…………………………………………………………………………………….35

**Some page numbers may not correspond appropriately due to removal of proprietary

information**

Abstract

Tissue-engineered systems involve the use of cells, growth factors, and scaffolds to repair

and restore tissue function. Ultrasound is used as a treatment for clinical fracture repair.

Evaluating the mechanism behind the efficacy of low intensity pulsed ultrasound for fracture

repair has revealed that the existence of a small mechanical force that may be beneficial to

repairing musculoskeletal tissues using tissue-engineered scaffolds. Using the elements of

scaffold synthesis, ultrasound, and principles of musculoskeletal tissue regeneration, a fracture

repair device is designed.

The proposed device is a bone fracture repair system that works with a scaffold

component as well as an ultrasound component. This device is designed to enhance current

techniques of bone formation in a more efficient and a minimally invasive way. It will serve as

an alternative solution to surgical methods of healing bone fractures, especially since the medical

industry is shifting towards least invasive methods of treatment. This device will also serve as

the answer to healing non-union fractures, or fractures that do not heal on their own for unknown

reasons.

Compared to other systems in the medical industry, this project is low-cost and has

research proving its future success. Recent data has proven that a low intensity ultrasound beam

mimics the effect of mechanical loading on bone formation. SolidWorks simulation of the

viscoelastic deformation in response to a mechanical force will serve as the preliminary sketch

for designing parameters of the system. This proposed system holds the potential to revolutionize

orthopedics by becoming the new standard in non-union fracture treatment.

1 Introduction

1.1 Background (Client and Disability)

In the field of biomaterials, researchers and clinicians are constantly looking to improve

the efficiency and rate of healing of fracture repair. It is shown for non-union fractures that all

periosteal and endosteal repair processes will stop if there is no surgical intervention. Therefore,

it is convenient to both the clinician and primarily the patient, to have a less invasive, alternative

means for treatment. Delayed healing can lead to various complications such as infection,

malformations of the healing site and a multitude of secondary defects.

The most common method of bone repair consists of resetting the joint or bone to a

position of function, immobilizing it there and simply waiting for the natural healing cycle to

occur. Up to 10% of fractures can be categorized as delayed-union or nonunion, both of which

require secondary intervention [1]. These interventions usually require the bone to be connected

to itself using a plate and bone screws. In some cases, improperly applied primary treatments

result in nonfunctional healing processes such as hypertrophic non-unions.

Dr. Khan is a professor of Biomaterials at the UConn Health Center. His work with

biomaterials has focused on orthopaedic devices and how they can be improved. Through his

research, Dr. Khan has proven that osteoblasts form mineralized bone tissue faster when

subjected to Low-Intensity Ultrasound (LIPUS). He would like a system to be designed that

includes the necessary parts for a clinician to seed a scaffold with osteoblasts and then inject it at

the desired wound site. This can then be activated with an ultrasound in a procedure that is

minimally invasive.

1.2 Purpose of the Project

The purpose of our project is to design a non-union fracture repair device that will

minimize the negative effects of current fracture repair methods. Our biomaterial scaffold will be

constructed to create a three-dimensional (3D) environment. When designing a tissue

engineering system, the choice of biomaterials that will be chosen for the scaffold must also be

considered.

As mentioned previously, there are many negative effects to current bone tissue healing

methods. Existing methods consist of autografts, allografts, and xenografts. With regard to

autografts, the negative consequences include donor site morbidity, limited tissue supply,

invasive surgical approach and risk of infection. The shortcomings of allografts and xenografts

are immunogenicity, disease transmission, length of lifetime variation, graft-vs.host-disease, and

the potential need for immunosuppression. Because of these deficiencies, there is a great demand

by clinicians to have a less-invasive means that will reduce the risk of infection during fracture

repair.

1.3 Previous Work Done by Others

Dr. Julius Wolff developed Wolff’s Law which states that bone is formed and resorbed in

response to the stress applied to it. Bone responds to mechanical stimulation where a load

induces bone formation and lowered load induces bone resorption. This is an important concept

to understand while considering fracture repair since bone formation is key to healing at the site.

It has recently been discovered that low-intensity pulsed ultrasound can be used to stimulate the

osteoblasts of injured bone when mechanical loading is not possible.

Ultrasound has historically been used for therapeutic soft tissue treatment, imaging and

tissue destruction. However, recent results have shown that decreasing the intensity in ultrasonic

beams can initiate cell proliferation, particularly osteoblasts and osteocytes. These devices

transmit sound pulses into the body using a probe and are composed of four main parts. An

electrical signal is sent through a transducer, which converts the signal to low amplitude

mechanical vibrations. Next, the generator uses its electrical energy to convert these vibrations to

vibrational energy by a transducer that contains a piezoelectric element. A low-intensity

ultrasound uses lower intensities to prevent damage to tissues, as well as to provide a stimulus

for cell proliferation.

Researchers have also learned how cells react to the administration of low-intensity

pulsed ultrasound throughout the four stages of fracture repair. These stages are inflammation,

soft-callus formation, hard-callus formation, and remodeling. It has also been proven in in vitro

cell studies that low-intensity pulsed ultrasound exposure triggers the activation of hypoxia-

inducible factor-1a and an increase in nitric oxide production. This leads to the increased

expression of growth factors in osteoblasts which stimulates angiogenesis. Angiogenesis is the

development of new blood vessels and is a key component in not only early bone repair but also

as a necessary precursor to endochondral ossification [1].

1.3.1 Products

Pins, plates, nails and screws are the currently used for fracture repair where bone union

wouldn’t otherwise occur on its own. However, these products all must be placed during open

surgery, and no minimally invasive system has been proposed such as an ultrasound - mediated

scaffold device system.

DBX Bone Graft Substitute is a commercially available product that is that consists of

demineralized bone matrix from human donors in a biocompatible carrier that is fully degradable

in six months [4]. The bone matrix is made of collagen and bone morphogenic proteins (BMPs),

a type of bone growth factor. The carrier is sodium hyaluronate formed by plasma membrane

proteins. This is injected into the bone fracture site and can be used to fill bone voids or gaps to

promote bone growth, but cannot be used where mechanical stability is important to the bone

structure. DBX must be used in conjunction with bone fixation devices as described above in

order to maintain mechanical integrity. Our proposed design system will not only promote

osteoblast proliferation and growth, but also should be able to retain mechanical integrity without

invasive procedures involving fixation devices.

The CMF OL1000 Bone Growth Stimulator [5] is the closest product on the market to

our proposed design system. This is a treatment system designed for use with non-union

fractures, similar to the goal of our design. However, this product uses an electromagnetic waves

as its stimulator as opposed to ultrasound. The CMF OL1000 Bone Growth Stimulator can be

used with internal or external fixation or over a cast, while our design will have an injected

scaffold that will harden in the body and should eliminate the use of fixation devices.

1.3.2 Patent Search Results

United States patent number 7510536 is similar to the proposed system device, but this

concerns the use of high - intensity ultrasound for use with nerve damage repair. It is held by

Foley et al. and was issued on July 3, 2012.

Additionally, patent number 8,057,408, issued to Cain et. al on November 15, 2011,

identifies a procedure of using pulsed cavitation ultrasound as therapy for bone repair, but as the

proposed device will not be using cavitation, this is not a concern.

Patent 8,226,582 outlines the process of using a modal converter assembly used to deliver

acoustic energy to a tissue sample to induce a healing effect. Specifically, the transducer is

described as being perpendicular to the tissue surface and delivered as both shear and

longitudinal waves.

The NELL-1 patent (8,207,120) was issued on Jan 26, 2012 and refers to an osteogenic

polypeptide (NELL-1) that is has very favorable bone mineralization properties, and can be used

as components of bone grafts or as a “stand alone agent” for enhancement of fracture repair.

Patent number 8,123,707 refers to a procedure for therapeutically treating connective

tissue or increasing vascularization in tissue using ultrasound. This patent was issued February

28, 2012 to Huckle, et. al. This product focuses on treating spinal discs only, and the proposed

device will use a similar pulsed ultrasound procedure, but aims to target in conjunction with a

stabilizing scaffold in the wound

However, these models are all significantly different than the proposed device and thus

patent infringement is not an issue.

1.4 Map for Rest of Report

Future work includes developing our project design and expanding upon our optimal

design. Realistic constraints and safety issues that pertain to our project will also be discussed in

the report. The impact of our design as a solution to healing non-union fractures and its future

use to clinicians is a critical component to the purpose of our design. Thus, we will further

explore the global impact of this solution and how it can be utilized for a variety of fracture

repair applications.

Although this project is in its initial stages, we are learning to collaborate as a team.

Communication skills have proven to be of extreme importance, as well as learning to

thoughtfully listen to what each partner has to say. As the project progresses, so will our life-long

learning.

Budget and timeline progression will be updated with each passing week. This is to

ensure that certain deadlines are being met. It will also be useful in designing a scaffold that is

comparatively inexpensive. This will hopefully make the finished product more appealing to

marketing companies, as well as insurance companies to cover the treatment.

Each team member is critical to the success and progression of the project. Contribution

and input from every member allows us to move forward and be as efficient as possible. The

final report will contain a section dedicated to describing each team member’s contribution to

our final product.

2 Project Design

2.1 Introduction

In order to develop a method to enhance bone growth at fracture sites, several designs

were proposed. From them, one optimal design was chosen and modified for the fracture repair

system. This design not only encourages osteogenesis, but is also non-toxic and biocompatible.

2.1.1 Alternative Design One: PLGA Scaffold

One method of design incorporates PLGA or specifically LGA polymer (PLGA 8515

DLG 7E, Mw = 120 kDa, Mn = 97 kDa) into the scaffold. Polymer scaffolds are considered an

option to due to their decent biocompatibility and chemical versatility. There are no dangers of

immunogenic responses or any possibility of disease transmission. Additionally, to act as a

synthetic matrix, the structure and surface morphology of the scaffold has to meet general

requirements. An active blood vessel network is required for cells to survive and integrate with

existing host tissue. Interconnected pores are necessary to ensure cell growth as well as flow of

nutrients and metabolic waste transport. The scaffold shape should facilitate cell seeding and

attachment.

In our design, we would prepare the scaffold by the solvent/casting particulate leaching

method. Salt (NaCl, Mw 58.443 g/mol, solubility in water 36g/100mL at 20 °C) and 1,4-Dioxane

are both used in preparation of the PLGA scaffold. 700 µL of PLGA solution (15% w/v in 1,4-

Dioxane) would be cast drop by drop into Teflon molds filled with 700 mg of NaCl porogen

particles with 600–1180 µm diameter. The mold containing the porogen and the polymer solution

would be first maintained at room temperature overnight to permit the diffusion of the polymer

solution through the porogen particles, and then placed at −25°C for 24 hours. The frozen

porogen/polymer mixture would be freeze-dried at −50°C for 12 hours to completely remove the

solvent. The scaffolds were dialyzed in water (200 mL) at room temperature for 21 days to

remove the porogen particles. The water was changed three times a day for the first week and

then once a week. After dialysis the scaffolds were freeze-dried at −50°C overnight. The

prepared scaffolds were stored in a dessicator at −25°C. The scaffolds have a height of 6 mm and

a diameter of 12 mm [6].

After creating the PLGA scaffold, the entire fracture repair system will also utilize an

ultrasound pulse to promote cell proliferation. In the initial stage, we will use alginate beads to

model cell activity. However, it is important to note certain drawbacks. A fundamental problem

with using these polymers is their degradation and the subsequent release of the lactic acid and

glycolic acid monomers would cause the pH of the surrounding extracellular fluid to drop and

interfere with the mineralization of bone. The acidity has been implicated in both the catalysis of

further scaffold degradation and the eliciting of a pronounced inflammatory response leading to

inhibition of tissue formation. Additionally, a PLGA scaffold is more rigid than the other options

we are considering. This is unsuitable for the noninvasive technique of injecting the scaffold at

the fracture site. Overall, a PLGA scaffold has beneficial aspects towards the goal of the design

project. However, there are also many shortcomings in this alternative design, and there are

several other material options that would be more appropriate for use with bone fracture repair.

2.1.2 Alternative Design Two: Hydroxyapatite scaffold

A bone substitute called hydroxyapatite has shown positive clinical results in terms of

osteogenesis. Hydroxyapatite (HA) is a mineral with the chemical formula Ca5(PO4)3(OH).

Most implants are currently coated with HA in order to promote bone growth and enhance the

biocompatibility of the device. Present studies depict hydroxyapatite to be used as bone matrices

for specific release as well as bone regeneration.

Due to the fact that support is one of the main factors that needs to be addressed when

trying to enhance fracture repair, hydroxyapatite would be a good material solution for this

situation. It has almost the same chemical makeup as bone, and promotes osteoinductivity,

giving the surrounding tissues mechanical stability.This is important so that the existing bone is

held in the proper position, ensuring that new osteoblast and osteoclast activity is occurring in

the correct areas during the healing process. Additionally, hydroxyapatite has great bioactive

and osteoconductive properties, thus making it a good material for scaffold design.

Hydroxyapatite scaffolds can be functionalized with molecules (collagen, chitosan, etc.), to

enhance their biological response.

A hydroxyapatite scaffold would involve an inner structure with interconnecting pores. In

order to promote vascularization, the pore diameter must be greater than 300µm. These pores

will then be large enough for effective bone cell attachment [7]. Porous hydroxyapatite can be

used for drug delivery and to further enhance nutrient transport and tissue infiltration. The pores

that we will be creating will have diameters of 500µm in order to maximize the space available

for new tissue generation.

There are several techniques that can be used to meet the criteria for the porous

hydroxyapatite scaffolds discussed above. These techniques include the use of gel casting, gas

foaming, slip casting, freeze casting, fiber compacting, and solid free-form fabrication. Other

methods such as polymer replication, give rise to the possibility of fabricating and designing a

custom scaffold with controllable pore size [8]. The method that we will be using to design our

porous scaffold is a technique known as polymer sponge templating.

Through polymer sponge templating, we are able to design a porous, interconnected

scaffold. This method of replicating a porous polymer template allows us to have control over

the pore size for that scaffold that we are constructing, as well as achieve the desired shape and

orientation of the pores. One limitation to this method however, is that our scaffold will have

difficulty sustaining high load-bearing applications [9]. Nevertheless, because we are able to

customize our porous scaffold design, this method proves to be most efficient for

vascularization. The ceramic hydroxyapatite will need to be synthesized with strong mechanical

properties that will compensate for the weakness in our scaffold as a consequence of customizing

the pores.

The HA will be synthesized using a precipitation technique. We will start with Ca(NO3)2

4H2O and a distilled water-H3PO4 solution of pH 5. Ammonia is used to order to get the proper

acidity. The 0.5M Ca(NO3)2 4H2O will be added to the solution next, maintaining a Ca/P ratio

of 1.67. The precipitates will be filtered out using a Buchner funnel, washed repeatedly using

double-distilled water to remove NH4 1 and NO3 and dried at 650 degrees C for 24 h in an oven.

Next, the powder will be ground up and then used [10]. The particle size will be identified using

a TEM.

The method of insertion of this scaffold will be during a surgical procedure, where the

patient will be required to be put under anesthesia. Because this is an invasive procedure, the

patient is prone to various pathogens and infection. Despite the biocompatibility and bioactive

properties of our porous hydroxyapatite scaffold, there is always risk of immunogenicity as well.

Hydroxyapatite has potential due to its bioactive properties and availability for ion exchange

with the surrounding fluid. However, hydroxyapatite is very brittle and would not deform

significantly when activated with the ultrasound. Ultimately this fracture repair system that is

being designed must eliminate the need for an invasive procedure, and in this case there is most

likely another material with similar biocompatible properties that could be used for this

procedure.

2.1.3 Alternative Design Three: Alginic Hydrogel Scaffold

Alginic acid, also known as algin or alginate, is a polysaccharide capable of forming a

lightweight hydrophilic matrix. This structure can be prefabricated or spontaneously formed

under suitable conditions. In both cases, the Algin hydrogel is capable of significant rates of ion

and solution exchange when placed in an aqueous medium. Algin is suitable as an implantable

biomaterial due to its high biocompatibility and low immunogenicity. Purified alginate is

typically manufactured from sodium-alginate extracted from brown algae. This substance is

both inexpensive and easily obtained.

Although Algin naturally degrades at a very slow rate in neutral pH systems, it can be

manipulated to degrade faster or slower by manipulating factors such as gamma-exposure or

partial oxidation. By controlling the amount of crosslinking present in the polymer, Algin based

hydrogels have been designed that degrade in less than 5 days and greater than 50. The

degradation products are non-toxic and typically cause no adverse effects at the implant site or

the rest of the system.

The hydrogel will closely resemble the extracellular matrix of soft tissues. If implanted

at a bone defect, the hydrogel will be too flexible to replace the physical functions of the missing

bone. However, it would be suitable for suspending osteoblasts and other cells needed for bone

formation. In addition to this, the relatively low Young’s modulus would allow for the necessary

deformation when exposed to low-intensity ultrasound waves [7].

To form the base Alginate solution, Alginate powder is dissolved in deionized water.

This solution is then combined in equal parts to a solution of methacrylate-anhydride and then

titrated with Sodium Hydroxide over the course of 72 hours. The resultant solution is combined

with ethanol to form a precipitate and then filtered. The retentate is then dissolved in deionized

water to be precipitated and filtered a second time. The resultant retentate will be a slurry of

water and methacrylate-alginate [11]. This will be stored wet at 37ºC in an airtight container to

prevent dehydration induced crosslinking.

Prior to implantation, this solution would be mixed with a sample of the intended

patient’s own osteoblast cells. The resultant solution will be a saline suspension of osteoblasts

contained within the cross-linked alginate matrix. The saline solution would have an osmotic

pressure slightly lower than that of human extracellular fluid. This would promote ion and solute

flux into the hydrogel upon implantation, and thus promote faster bone healing by providing the

osteoblasts with necessary ions as soon as possible.

The osteoblast and alginate suspension would be loaded into a syringe and implanted

directly into the wound or defect site via injection. This procedure would be minimally invasive

and easily performed with only an ultrasound imaging device to guide the surgeon. Single-use

syringes and sterile manufacturing conditions for the hydrogel would greatly reduce chance of

infection. Alginate hydrogels are known to be effective as calcium ion delivery vehicle, however

it has not been very well tested or documented for usage as anything other than a drug delivery

system, and we do not have the time or the resources to research the properties of the material. In

this case, it would most likely be a better idea to use another material with known biocompatible

properties that could be used in a hydrogel to be stimulated by the ultrasound.

2.2 Optimal Design

Content removed to preserve proprietary data and information, this information

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2.2.1 Objective

Figure 1: Anticipated workflow

Overall, the scaffold that is being created is made to be injected into a fracture site to

harden when exposed to the body environment, providing mechanical strength. The setup of the

completed fracture repair system is shown in Figure 3, with the cells seeded onto the scaffold,

injected at the fracture site. The pulsed ultrasound wave is transmitted through the skin to

stimulate the proliferation of the cells on the scaffold. Our design includes the scaffold, which

will then be used as a component of a fracture repair system.

In order to develop a scaffold that has biomimetic properties, we will be modeling the

viscoelastic behavior using a mechanics simulation suite called ANSYS. Viscoelasticity is a

property that combines both elastic and viscous properties to characterize the time dependent

strain and deformation based on an applied force. Once we construct the scaffold, we will test the

properties using an imaging system called Volocity to wave the movement of the fluorescent

bead markers when exposed to the ultrasound waves.

2.2.3 Controlled Stimulation

The device that will be used to stimulate osteoblast proliferation and differentiation at the

fracture site is a low-frequency ultrasound. The cells will be seeded on the scaffold prior to

injection at the site. Ultrasound has historically been used for therapeutic soft tissue treatment,

imaging and tissue destruction. However, recent results have shown that decreasing the intensity

in ultrasonic beams can initiate cell proliferation, particularly osteoblasts and osteocytes. These

devices transmit sound pulses into the body using a probe and are composed of four main parts.

An electrical signal is sent through a transducer, which converts the signal to low amplitude

mechanical vibrations. Next, the generator uses its electrical energy to convert these vibrations to

vibrational energy by a transducer that contains a piezoelectric element. An amplifier is used in

order to send the correct intensity to the location. A low-intensity ultrasound uses lower

intensities to prevent damage to tissues, as well as to provide a stimulus for cell proliferation.

Figure 4 shows the ultrasound setup that we will be using to stimulate cell growth on the

scaffold.

Figure 4: The low-frequency ultrasound includes a waveform generator, power amplifier,

and transducer.

This device outputs waveforms such as DC, sine, square, triangle, positive and negavite

ramps. Most importantly, it has a frequency range of 0.001 Hz to 100 kHz. In this design project,

we will be making a scaffold, and testing its response when stimulated by pulsed sine wave

frequencies at 100kHz, 100 Hz, and 10Hz. This function generator is hooked up to a power

amplifier and the transducer.

Figure 5: The function generator being used is a PI-8127 from PASCO Scientific, as

shown above.

The transducer converts electrical energy into ultrasound, or sound waves. It does so due

to the piezoelectric element that is housed inside the transducer. When a voltage is applied,

piezoelectric crystals are able to change size and apply an AC current so that the crystals are able

to oscillate at very high frequencies, producing high frequency sound waves. The frequency

range that we will be dealing with when testing is between 100kHz and 10Hz, which could be

considered ‘low-intensity’. The transducer and ultrasound setup will be used daily when ready to

test the scaffold for osteogenic properties. The device will be pulsed for 20 minutes per day for

seven days.

The reason why we have chosen this time schedule to test our scaffold is because several

studies with positive cell proliferation results have exposed osteogenic cells 20 minutes per day

to a pulsed ultrasound [13]. Because this is a design project, we will not be researching the most

ideal exposure time of the stimulator in terms of increased osteogenesis. We will mainly be

testing for the best material for the scaffold, and finding the properties of that material, such as

the viscoelastic constant, based on the cell growth rates when stimulated by ultrasound.

Researchers have, however, already learned how cells react to the administration of low-intensity

pulsed ultrasound throughout the four stages of fracture repair. These stages are inflammation,

soft-callus formation, hard-callus formation, and remodeling. It has also been proven in in vitro

cell studies that low-intensity pulsed ultrasound exposure triggers the activation of hypoxia-

inducible factor-1a and an increase in nitric oxide production. This leads to the increased

expression of growth factors in osteoblasts which stimulates angiogenesis. Angiogenesis is the

development of new blood vessels and is a key component in not only early bone repair but also

as a necessary precursor to endochondral ossification [13]. Based on early evidence, the low-

intensity ultrasound should promote bone growth on the scaffold that we construct.

Osteoblasts are the cells that mineralize bone and at it to the preexisting structure. These

cells are matured from pluripotent osteoprogenitor cells. These osteoprogenitor cells are an

intermediate phase between mesenchymal stem cells and matured bone cells. Osteoprogenitors

have the potential to become either osteoclasts or osteoblasts based upon the environmental

conditions present. As a clinical device, our scaffold will be seeded with these osteoprogenitor

cells. A combination of bone morphogenic proteins and the applied low-intensity pulsed

ultrasound will induce differentiation into osteoblasts.

This differentiation from osteoprogenitors to osteoblasts is detectable by using a real-time

polymerase chain reaction to confirm that specific enzymes are being produced. This is a reliable

method of determining the rate and stage of differentiation because these enzymes are only

present at a certain ratio when the cells are osteoprogenitors and then another ratio as osteoblasts.

Jim Veronick, a researcher in Dr. Khan’s lab has already confirmed that the range of low-

intensity pulsed ultrasound we intend to use does have an effect a measurable effect on these

enzyme ratios. In order to make the final clinical device as versatile as possible, Dr. Khan has

requested that we characterize our scaffold in such a way that a viscoelastic equation can be used

to describe the deformation that will occur at different intensities of ultrasound exposure. This

equation will be a function of some unknown coefficient and the percent-weight composition of

the scaffold. Using this characterization, the scaffold will be tunable at a clinical level in order to

optimize the positive effects on osteoblast proliferation and differentiation based on the size and

location of the bone defect.

2.6 Integration of all these components into a device

This system will include the necessary devices to; harvest the necessary tissue for a

cellular osteoblast autograft, seed them within the scaffold, inject the seeded scaffold into the

defect site, and the transducer needed to activate the scaffold with ultrasound. Overall, the

package will be easy to use for any healthcare provider familiar with intracavitary injection and

cell fluid purification. The chemicals and materials used will be purified to the FDA standards.

In addition to this, the scaffold reagents will be sterilized to their individual pharmacological

requirement levels.

This system holds the potential to become the standard procedure for treating nonunion

and delayed-union bone fractures. The application of acoustic energy without the proposed

scaffold has already been shown to greatly decrease the healing time of a bone defect [1]. The

scaffold will allow for quicker infiltration of osteoblasts into the defect and even faster healing.

2.2.7 SolidWorks Simulation

Several SolidWorks simulations have been performed to determine how the proposed

device would operate and respond to ultrasound energy. The most basic design consisted of a

composite column being subjected to a single constant force on one of its faces. This column

had a cross sectional area of 100mm2 and a height of 52mm. The column was split into three

separate partitions to simulate the scaffold being sandwiched between two pieces of bone. The

bone segments were each 25mm tall and the scaffold segment was 2mm tall.

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Figure 7: Properties of the Design

Because these simulations were preliminary, and no data had been collected yet, the

different partitions were assigned the material properties of homologous polymers. The bone

segments were given the material properties of polyurethane and the scaffold was assigned the

properties of rubber. The simulations were designed only to determine the relative deformation

of the material in different locations, so the only important factor about the assigned material

properties is that the homologous bone polymer has a glass transition temperature above the

testing temperature (25ºC) and the homologous scaffold polymer glass transition temperature

was below the testing temperature.

The most basic simulation performed consisted of a single, constant force of arbitrary

force being applied to a single face of the model. The simulation was then run, and it was

analyzed to determine the resulting stress and displacement of the model. As shown below in the

stress diagram (Figure 8) and displacement diagram (Figure 9), the effect of a constant force is

not equally distributed throughout the entire volume of our assembly. These diagrams show an

exaggerated view of the affected areas, and are not true representations of how the model would

look under the same force. However, it is still significant that the terminal edges on the face of

the device are affected by the applied force much more than other areas. This suggests that

optimal application of ultrasound may require it to be applied from multiple angles to result in

symmetrical loading of the scaffold and thus the suspended osteoblasts.

Figure 8: Von Mises Stress Figure 9: Displacement

3 Realistic Constraints

This design accommodated engineering standards by adhering to FDA guidelines

regarding laboratory protocol as well as testing procedures for biological material. The scaffold

was also modified from its original design in order to comply with a more affordable, easy-to-

construct product and to attempt to eliminate hazardous waste with sustainability and the

environment in mind, as well as complying with ethical guidelines.

Economically, our device is low-budget due to the fact that we will not initially be using

live cell culture samples. The ultrasound setup itself costs several thousand dollars, however this

is available to us free of charge as well as laboratory space, and can be used an indefinite number

of times. Depending on the rate at which we are able to finish the design, we may be able to

proceed to seeding osteoblasts onto the constructed scaffold.

The finished system itself will prove to be very economical as well. The cost of the

scaffold and injection will be much less than that of an invasive surgery, where one must

consider the cost to run each piece of equipment, the salary of each person in the operating room,

the price of the facilities and the cost of the stabilization devices themselves. The ultrasound

machine will be able to be used multiple times for multiple patients several times per day, and

the scaffold itself will be constructed of a very affordable and available biopolymer. Overall, not

only is the design itself low-budget, but also the system that will eventually be constructed

involving the scaffold is economically affordable. In terms of environment and sustainability

issues, one of the materials that we will be using in the testing of our scaffold is red fluorescent

polystyrene microspheres. These will be used as markers when looking at the viscoelastic

properties of the scaffold. Polystyrene is chemically inert, and is resistant to acids and bases. Due

to this non-biodegradable characteristic, waste disposal of this polymer must be taken into

consideration and factored into costs. Additionally, polystyrene is made from petroleum, a non-

sustainable, non-renewable resource. There is such a thing as ‘polystyrene recycling’, but this

would not apply to us because the scaffold will be mixed with the fluorescent polystyrene beads

when the materials would need to be disposed. However, the fluorescent markers are only used

during the testing of the scaffold to determine the properties such as the viscoelastic constant,

and will not be used during injection into the body. Because the polystyrene will only be used for

in vitro testing of the design, the sustainability concern here is not too impractical, and very little

waste will be generated as a result.

The manufacturability of the finished system is very feasible. Regarding manufacturing

constraints, the scaffold needs to be developed in a “clean” environment, as approved by the

FDA. Because this scaffold will be designed with the intention of being injected into the body,

this is an important consideration during the design process. The FDA states that for devices of

biological origin, the methods and details of the sterilization process and validation through

mechanical testing data be submitted in an IDE (Investigational Device Exemption), and these

must follow the AAMI (Association of Medical Instrumentation) guidelines. Additionally, from

a manufacturing standpoint, the storage procedure and shelf life is also an important constraint.

According the the FDA, the shelf life should be stated with data accompanying the device

demonstrating that the properties are not deteriorated with long periods of storage. Therefore, the

scaffold must be designed with this in mind.

In terms of ethical constraints, there are very few as well. After the development of the

scaffold, we will potentially be seeding cells onto the scaffold before inserting it into the body.

The osteoblasts are from mice, but are not harvested in Dr. Khan’s lab, but are purchased in the

form of a cell line. The method of regenerating more cells from previous cells is not only

economical, but it eliminates the unnecessary killing of many mice. In fact, one vial of cells can

sustain a lab for five to ten years, and so this should not be an ethical concern during the project.

The UCONN Health Center pays for proper hazardous waste disposal for Dr. Khan’s lab.

The solvents that are used in the process of creating the design scaffold are not carcinogenic, and

therefore this is not a concern. However, in terms of the overall system, the waveform generator

must not be disposed of in a standard waste container, as dictated by the European Union WEEE

(Waste Electrical and Electronic Equipment) symbol on the generator. The amplifier also must

be disposed of with electrical waste, and the cables that are hooked up to the transducer. The

disposal is important in terms of ethical issues because although the UCONN Health Center pays

for proper waste disposal, we are not personally ensuring the correct procedure is taking place as

we are trusting the employed professionals in this matter.

In terms of health and safety, biocompatibility of the scaffold is the main concern in this

design. As stated earlier, our product is meant to be used to avoid the negative effects of

traditional bone regeneration methods. Therefore, it is critical to ensure the health and safety of

the patient and refrain from disease transmission or immunogenicity.

Politically, our healthcare system appears to be going through a period of reform, and

there are two aspects that pertinent to our design and overall plan to create a clinical device..

First, it must be feasible for companies to produce and market the medical device system.

Second, the overall fracture repair system must be able to be covered by health insurance

companies or else the product and it’s positive effects will be negligible. The Medical Device

Excise Tax that went into effect on January 1, 2013 will affect the production and manufacturing

of the clinical system. This institutes a 2.3% tax on the first sale of medical devices with an

exception of eyeglasses, contact lenses, hearing aids, and other devices that are generally

available and purchased by the public at retail. This would mean that the fracture repair device

that is the overall goal of the client, Dr. Khan, would fall under this new tax when trying to get a

company to market and produce the new medical product. This is one constraint to consider, as

we must make the scaffold very affordable so that companies will still find the appeal of such a

system, regardless of the added tax. With the healthcare reform and the restructuring of insurance

companies, another concern is if the average American will even be able to afford using a system

such as this one, and if these costs will be facilitated by medical insurance. Fortunately, because

this is a minimally invasive system, we are eliminating the astronomical costs of typical surgery,

and the ultrasound machine, which is the most expensive portion of the system, can be reused

many times per day, thus making this an economical option that most insurance companies

should be willing to cover, as theoretically, a treatment such as this one would save them much

more money than the usual fracture repair surgeries.

Overall, there are several design restraints that were taken into account for this design,

but most, if not all, are so minor that the construction and testing of this design should go as

planned.

4 Safety Issues

The health and safety aspect of this design can be examined from the position of the

patient, the people working on the project, and the clinician. Biologically, the most important

characteristic of the scaffold to be constructed is its biocompatibility. Because the main objective

is to inject the scaffold at the site of fracture, the scaffold must not have an adverse reaction

when in the body. In fact, the host reaction to the biomaterial will theoretically result in increased

osteoblast proliferation and differentiation. This is important because the completed bone

fracture repair system aims to correct some of the negative effects of surgical fracture repair

methods. Therefore, it is critical to ensure the health and safety of the patient and refrain from

disease transmission or immunogenicity. In fact, this design has less of an immunogenicity risk

for the patient than traditional methods such as allografts. Here, we are using immature

osteoblasts seeded onto the scaffold and not cells from another human. The real challenge is to

find a material that is biocompatible.

Safety for laboratory team working on the scaffold itself is also a vital aspect of the

design process.While constructing the scaffolds, disposable pipet tips will be used when mixing

the chemical reagents to create the hydrogels. This eliminates the possibility of mixing

substances into a toxic mixture. Personal protective equipment, such as gloves, will be worn at

all time when working with the chemical substances. Additionally, we have attended a UCONN

Health Center Biological Health and Safety class to become aware of the proper waste disposal

methods and chemical protocols in the facility.

One more safety concern to factor into the design is the possibility of the heating in tissue

due to the ultrasound. Dr. Khan’s laboratory team has already done tests of temperature increase

of saline solution to minim the body under the ultrasound intensities that we will be using. There

was less than 1 degree Celsius increase under these condition, which is well under the FDA

requirements. There are no risks currently found being around the ultrasound equipment, which

is important when considering the safety of the clinician who will be exposed to the device for

several hours at a time.

5 Impact of Engineering Solutions

The development of the non-union fracture repair system has many implications for

potential applications in the future. It is possible to use the same concept and design parameters

for other healing portions in the body. The proposed device develops a scaffold that will allow

optimal cell activity specifically geared towards healing. Upon developing the system, the

various parameters for creating the scaffolds can be specialized to different types of tissues. For

instance, a possible future development involves a similar system for blood vessel repair. The

benefits of our design allow it to be flexible towards different systems in the body.

The non-union fracture repair device would be utilized by clinicians for fractures that fail

to heal spontaneously due to injury. This system could be used in a hospital setting to improve

the efficiency and rate of healing of fracture repair. It narrows down the procedures necessary for

healing a fracture at various sites of the body. This standardization will be useful in the future

allowing physicians to have a direct solution with fracture repair. The objective in treatment is to

achieve bone union as early as possible.

The global impact of this solution can be interpreted with a variety of applications.

Fracture repair, as well as just general tissue repair is a concept that applies to humans all over

the world. Providing Dr. Khan with this fracture repair system will also provide a solution to

healing non-union fractures. It is possible for people to be faced with this problem of non-union

fractures all over the world. This is a solution that has the ability to improve the lives of those

suffering from non-union fractures and left with very limited options, including invasive surgery.

This will be an effective system that can have a significant impact on global solutions in the

medical world.

The economic impact of such a device is especially important to consider because this

device is well within a reasonable cost range when comparing to other medical devices in the

same field. Additionally, this method has been shown to be minimally invasive when it comes

down to the actual application. Other options would involve surgical intervention, which not

only increases costs, but also brings a decrease in the efficiency of healing time. The clinical

costs would be exponentially higher versus the simple design of an injectable scaffold that will

decrease the amount of time necessary for fracture repair. This is especially important because

having the most efficient solution when treating these non-union bone fractures allows progress

to developing even more efficient solutions.

The environmental impact of such a solution is most definitely in a positive manner.

There are no hazardous materials involved, and everything is created to be biocompatible. This

kind of solution is better for the environment since there is increased efficiency as well as no

toxic waste. The materials involved can also have multidisciplinary applications which allow

future progression to occur. Since the materials are known for their various properties, different

perspectives on the design can also help developments.

The societal effects involve the effects of implementing this design. In the field of

biomaterials, researchers and clinicians are constantly looking to improve the efficiency and rate

of healing of fracture repair. It is shown for non-union fractures that all periosteal and endosteal

repair processes will stop if there is no surgical intervention. Therefore, it is convenient to both

the clinician and primarily the patient, to have a less invasive, alternative means for treatment.

Delayed healing can lead to various complications such as infection, malformations of the

healing site and a multitude of secondary defects.

6 Life-Long Learning

It is interesting to note that this design concept of a scaffold system used in collaboration

with ultrasound pulses can also apply to other forms of tissue repair. We are determining

parameters of a system that can remotely heal tissues with an implanted structure. The idea is

using acoustic energy and a compatible scaffold to initiate cell activity. This is made possible by

tweaking the parameters of the system. For instance, the viscosity of the scaffold can be altered

to work with the different types of tissues. This is especially exciting because we will have

developed future efficient solutions for tissue repair in a minimally invasive manner. This can

revolutionize options for patients in the clinical setting.

We also were fortunate to learn the inner workings of Dreamweaver for developing the

website that will be maintained throughout this project. This is especially useful in the future

because being able to organize a website is impressive in presenting various ideas to the outside

world. Another tool that has greatly increased our efficiency in completing the various reports

for Senior Design was Google Docs. It is especially great because all the group members can

continue to collaborate outside the classroom or group meetings. We can actively see everyone’s

input and discuss our ideas. The active editing process is also helpful because we can all witness

where everyone is coming from when they add a portion. This will continue to be helpful in the

future because actively collaborating with your group will keep you updated on your group

situation.

Although this project is in its initial stages, we are learning to collaborate as a team.

Communication skills have proven to be of extreme importance and learning to listen to what

each partner has to say. Being able to effectively communicate is an important life skill because

it teaches us as a group to deal with various issues in a professional manner. As a team, we are

also learning to realize and encourage the different strengths that each member is able to provide.

Another important life lesson with regard to senior design is being able to understand each

member’s vision of the project. This has allowed us, as a team, to create one optimal vision of

the project we wish to construct. Managing our time and structuring our schedules to meet

specific deadlines has helped our team focus on the task at hand, suggest alternative designs, and

clarify the final optimal design based on a team decision. The decision for the optimal design

was made with the client’s best interest in mind. In the short amount of time spent together as a

group, working as an efficient team has increased our workpace and lessened the possibility of

risks or mistakes.

Therefore, senior design will provide us with the necessary tools to succeed in the future

as professionals whether we are in industry, research, or the medical field. It is imperative that

we are able to effectively communicate to our colleagues and collaborate in an efficient,

respectful manner with one another. As biomedical engineers, many real-world situations will

require teamwork and the ability to work with others. Discrepancies and other issues must be

handled in a professional manner without degrading any of the team members. Each member and

their input is extremely valuable and should not be discouraged. Working together as a team

willing to listen and understand one another, we can provide the most optimal product for our

client.

7 Budget and Timeline

7.1 Budget

Overall, this can be considered a low-budget project. According to the client, everything

necessary for this non-union fracture repair device can be created for under $10,000 easily. This

is especially important to note since similar medical devices are usually at a higher price range.

There are other systems similar to this proposed design currently on the market that are

around the $10,000 cost level. For example, Symphony II Platelet Concentrate System has a list

price of $9,995.00 and concentrates the body’s natural growth factors which are found in

platelets. This is similar to the proposed device because Symphony II is a system that contains

the syringe needed to extract blood, and then the centrifuge needed to separate out the growth

factors, while our design is for a system that consists of the scaffold that is to be injected and

then the ultrasound to stimulate osteoblast proliferation. Another device that is similar to ours is

the CMF OL1000 Bone Growth Stimulator, which uses electromagnetic waves to promote

fracture repair, but this device can be sold for around $3000.00. This, however, has no growth

factors and must be used in conjunction with fixation devices which could cost upward of

$10,000.00.

The exact cost of the supplies is not yet known at this point. Access to the necessary

laboratories will be available at no cost. The ultrasound used in the design is also available free

of cost, as well as EHS Biological Health and Safety classes necessary for safety accreditation

when handling biological samples.

Fortunately, we were able to calculate the cost per use of the ultrasound which comes to

$2/hour. This was determined based on the fact that ultrasounds usually last 10 years and can be

used approximately 200 times each year. Thus, our total budget is reduced to $700.

7.2 Timeline

Content removed to preserve proprietary data and information

In order to see the timeline for this project, please refer to the report posted on the secure

website.

8 Team Members’ Contributions to the Project

Kelly Stratton

First, Kelly performed background LIPUS research in order to learn more about the

concept of using ultrasound in tissue regeneration. She also constructed a diagram of the fracture

repair system, including the parameters, and made a gif video to help the audience to better

understand the procedure. Kelly also compiled the products list, and found the costs of each

material. She then created the Microsoft Project file, updating it each week to accommodate for

more tests and restrictions. Additionally, Kelly researched the realistic constraints and wrote

three pages describing all the possible sources for constraint with this project. She created a flow

chart containing all four parameters that are to be tested, so that performing the experiments

would be simple and clear. Kelly regularly updated the website each week, and participated in

weekly meetings with the client at the Health Center. She also completed the Health and Safety

Training, as required by all faculty and students in the Health Center laboratories. Finally, she

engaged in background rheometer research, background Volocity software research, and

SolidWorks research on the displacement tests in order to understand these programs and easily

identify their outputs for relevant data. Kelly constructed and tested scaffolds on the rheometer.

Most of her time spent at the Health Center has been engaged in experimentation with the

various parameters on the rheometer in order to get consistent data regarding the elasticity of the

gels she has constructed. Over the semester, Kelly met with the team regularly to work on the

project statement & specifications, proposal, alternative designs, optimal design, and the final

report.

Yvonne Nanakos

Yvonne began by conducting a background search on osteoblast proliferation and

differentiation. This was done through various documents searches, which also included papers

given to the group by the client. It was important to understand which parameters were suitable

regarding the LIPUS to use in the design of the non-union fracture repair device. The design

process itself and background as to why our product is necessary was also done by Yvonne. She

also researched hydroxyapatite as an alternative design. In order to begin, Yvonne completed the

Health and Safety Training at the Health Center. This was required in order to work in the

laboratories of the University of Connecticut Health Center. With regard to the final

presentation, Yvonne completed a flow chart which outlined the design process of our product

and served as a visual representation for the project. It demonstrates components of the non-

union fracture repair device system, as well as the design methods the team will be using. The

conclusion for the final presentation and part of the background was also completed by Yvonne.

With regard to each writing assignment that was due (project statement, proposal, optimal

design, and final report), Yvonne equally contributed to the completion of each assignment. The

technical details and revision of the Final Report and Final Presentation were also done by

Yvonne. Finally, Yvonne has been to each weekly meeting with the group’s client at the Health

Center to discuss the progress made each week. In addition to the this work done, Yvonne has

also constructed nine scaffolds within the Health Center with the protocol from James Veronik.

Information regarding these gels were then tested on the rheometer. Yvonne spent time

researching the rheometer and learning how to load the gels appropriately.Yvonne tested 0.1%

and 0.2% gels on the rheometer and experimented with the various parameters on the rheometer

in order to become accustomed with this new piece of equipment.

Aimon Iftikhar

Aimon worked on background research for the methods of mechanical stimulation of

bone cells. She also researched concepts on Volocity and LIPUS while helping to configure the

realistic constraints. She researched various requirements of a fracture repair system and helped

determine the optimal design for the project. She worked to compile a GIF video that

demonstrates the completed design process of an injectable scaffold with the ultrasound pulses.

Additionally, she worked on researching and describing the polymer alternative design. She

helped in the website configurations of both the public and secure versions, including

troubleshooting errors to get the website in working order. She also researched the impact of

engineering solutions and lifelong learning as well as future directions of this project. She also

met with the rest of the team regularly to work on the various reports due this semester, including

the project statement & specifications, proposal, alternative designs, optimal design, as well as

this final report. Moreover, Google Docs was utilized to save these reports and be simultaneously

updated as each member worked on the reports. This allowed everyone to constantly see where

and what everyone was working on. Lastly, she wrote the script for the final presentation as well

as added technical details to the presentation. She completed the required Health and Safety

Training session at the Health Center. This is required, in order to be allowed to work in Dr.

Khan’s laboratory. She also learned the protocol for creating the scaffolds from James Veronick.

She also attended weekly meetings with the group and client to discuss progress as well as any

shortcomings or problems that may have arisen.

Nicholas Calistri

Nick Started the project by meeting with the client to discuss exactly what they needed.

In order to design an appropriate device, he performed research on standard Orthopaedic

practices, usage of LIPUS in clinical settings and modern fracture repair solutions. He then

worked to design an system using the Ultrasound as requested. He constructed a preliminary

flowchart to describe how the team would address the different components of the device. He

researched hydrogels, ceramics, and polymers in order to evaluate the optimal material for the

device. Once it was decided that we would be working in Dr. Khan’s lab, he attended the Health

and Safety Training course at the Health Center. He then investigated Abaqus, ANSYS, and

SolidWorks as possible resources to simulate our device. After deciding on using Solidworks, he

learned how to use the program to model and run motion simulations. He then designed five

unique models of fractures using physical characteristics and dimensions found in literature. He

also designed seven different motion simulations to learn how the injectable scaffold would react

to the forces of the applied ultrasound, gravity, and normal loading. In addition to this, he

performed multiple rheometer tests at the Health center different scaffolds.

9 Conclusion

The system we designed is meant to provide clinicians with a less-invasive means of non-

union bone fracture repair. The device will consist of a tunable scaffold which will be activated

by a designated frequency of acoustic energy. Healing fracture injuries with current methods

require a surgical approach and leave the wound prone to infection. With our design, we attempt

to minimize these negative consequences of standard bone tissue repair procedures (allografts,

autografts, xenografts). The system will optimize qualities of current scaffolds and meet the

requirements necessary for tissue repair.

When designing the system, we are faced with several limitations. Although our device

can be manufactured under a low-budget, other pressing issues (health, manufacturing, ethical

constraints) must be taken into serious consideration. With the proper awareness and caution

taken towards these limitations, we aim to create a self-enclosed device that includes all the

necessary components to seed a scaffold with osteoblasts and inject it into the wound site.

Following this, the wound site will be exposed to low-intensity pulsed ultrasound to promote

cellular proliferation and differentiation.

10 References

[1] Khan Y, Laurencin CT. Fracture Repair with Ultrasound: Clinical and Cell-based

Evaluation.J Bone Joint Surg Am. 2008 Feb;90 Suppl 1:138-44.

[2] Elisseeff J, Sharma B, Puleo C, Yang F. Advances In Skeletal Tissue Engineering With

Hydrogels. Orthod Craniofacial Res 8. 2005 April; 150-161

[3] Sharma B, Elisseeff JH. Engineering structurally organized cartilage and bone tissues. Ann

Biomed Eng 2004;32:148–59.

[4] DBX Bone Graft Substitute (2007). [Brochure]. MTF: Musculoskeletal Transplant

Foundation.

[5] CMF OL1000 Bone Growth Stimulator (2007). [Brochure]. DJO Incorporated

[6] Annalia Asti, Giulia Gastaldi, Rossella Dorati, et al., “Stem Cells Grown in Osteogenic

Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications,” Bioinorganic

Chemistry and Applications, vol. 2010, Article ID 831031, 12 pages, (2010).

[7] Karageorgiou, Vassilis, Kaplan, David, “Porosity of 3D biomaterial scaffolds and

osteogenesis.” Biomaterials, vol. 26, Issue 27, (2005): 5474-491.

[8] Texeira, S., Pena, R., De Aza, A.H., De Aza, S., Ferraz, M.P., Monteiro, F.J., “Physical

characterization of hydroxyapatite porous scaffolds for tissue engineering.” Materials Science

and Engineering, vol. 29, Issue 5, (2009): 1510-514.

[9] Ramay, H., Zhang, M., “Preparation of porous hydroxyapatite scaffolds by combination of

the gel-casting and polymer sponge methods.” Biomaterials, vol. 24, Issue 19, (2003): 3293-302.

[10] Gervaso, F., Scalera, F., Kunjalukkal Padmanabhan, S., Sannino, A. and Licciulli, A. ,

“High-Performance Hydroxyapatite Scaffolds for Bone Tissue Engineering Applications.”

International Journal of Applied Ceramic Technology, (2012): 507–516.

[11] West, Erin R., Min Xu, Teresa K. Woodruff, and Lonnie D. Shea. "Physical Properties of

Alginate Hydrogels and Their Effects in Vitro Follicle Development." Biomaterials 39th ser. 28

(2007): 4439-448.

[12] Cancan Xu, Wei Lu, Shaoquan Bian, Jie Liang, Yujiang Fan, and Xingdong Zhang, “Porous

Collagen Scaffold Reinforced with Surfaced Activated PLLA Nanoparticles,” The Scientific

World Journal, vol. 2012, Article ID 695137, 10 pages, 2012

[13] Function Generator: PI-8127. Roseville: PASCO Scientific, 2008. PASCO, 9.

11 Acknowledgements

We would like to acknowledge the following people with their help and insight on the

design project. Without their input, we would not have advanced as far as we have. Dr. Yusuf

Khan has provided valuable information about constructing the design and guiding us in the

entire process. Dr. Krystyna Gielo-Perczak has aided us in providing advice on technical details

of our design and presentations. Orlando Echevarria has been very helpful in troubleshooting our

public and secure websites. James Veronick has been extremely helpful in working with us on

the construction and testing of the scaffolds. Craig Hanna has provided helpful information about

the rheometer studies. Sarah Brittain has also aided us in the design technicalities.

12 Appendix

There were no alternative figures or diagrams used in this final report.