An Investigation into Osteoblast Adhesion on 3d printed scaffolds

Investigation of Osteoblast Adhesion on 3d printed scaffolds – Jemma Redmond – 09260340 – Msc Nanobioscience

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NanoBioscience Masters Project

“An investigation into the adhesion of osteoblasts on 3d printed scaffolds”

Jemma Jayne Redmond

Student Number: 09260340

Supervisor – Dr Brian Rodriguez Co Supervisor Dr Emmanuel Reynaud

Investigation of Osteoblast Adhesion on 3d printed scaffolds – Jemma Redmond – – 09260340 – Msc Nanobioscience

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Table of Contents

2.0 Acknowledgements ............................................... 3 3.0 Abstract ................................................................. 4 4.0 Introduction .......................................................... 5 4.1 What are osteoblasts? ........................................... 6 4.2 Their interactions with respect to bone remodelling 6 4.3 Stem cells .............................................................. 6 4.4 Collagen ............................................................... 7 4.5 Cartilage & bone formation ................................... 8 4.6 Tissue engineering ................................................. 9 4.7 Cell adhesion forces ............................................... 9 4.8 Creation of scaffolds – established methods ........... 10 5.0 Scaffold Materials .................................................. 10 5.1 Natural polymers ................................................... 11 5.2 Hydrogels .............................................................. 11 5.3 Designing the scaffolds through software 12 5.4 How the 3d printer operates .................................. 12 6.0 Materials and Methods 6.1 Batch preparation .................................................. 13 6.3 Preparation of cells for seeding onto platforms ...... 13 6.4 Scaffold Sample Preparation .................................. 14 6.5 Sample preparation for imaging ............................. 14 6.6 Design of the “S” samples ...................................... 14 6.7 Design of the “A” samples ...................................... 14 6.8 Design of the “B” samples ...................................... 15 6.9 The structure types that were used for the scaffolds. 15 6.91 Design of the D samples....................................... 15 6.92 Observed Osteoblast adhesion ............................. 17 7.0 3d printers ............................................................ 18 7.1 Software ................................................................ 18 7.2 – The bioprinter – a “phenotype” of the 3d printer 18 8.0 Pla versus abs ........................................................ 19 9.0 Results .................................................................. 19 9.4 How data was gathered ......................................... 21 9.5 Scaffolds – which were best? ................................. 21 9.6 Images and cell count ............................................ 23 9.7 Magnification ........................................................ 24 10.0 Discussion ........................................................... 24 10.1 Improvements made to the scaffolds ................... 24 10.10 The problems encountered in the creation of the scaffolds 27 10.11 The solutions developed in the creation of the scaffolds 28 10.12 On growing the cell culture on the scaffolds ...... 28 10.13 Imaging of the scaffolds – Problems with solutions 28 10.14 What could be done better next time? ............... 28 10.15 Other uses for pla implants ................................ 31 10.15 why do the cells not fit into the grooves? ........... 29 10.16 Ideas and method for the creation of new scaffolds 31 10.2 Improvements made to the 3d printer ................. 24 10.3 How could the process be improved? ................... 25 10.4 Straight Lines / Bridges ........................................ 25 10.5 Floating Samples.................................................. 25 10.6 What samples were the most effective? ............... 25 10.7 Limitations of the microscope .............................. 26 10.8 Printing of scaffolds through low cost 3d printing . 26 10.9 The materials used in the creation of the scaffolds 27 11.0 Summary ............................................................. 32


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12. 0 References .......................................................... 32

Appendix A ................................................................. 44 Appendix B .................................................................. 45 Appendix C .................................................................. 49 Appendix D ................................................................. 56 Appendix S .................................................................. 42 Appendix S(E)M........................................................... 41


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2.0 Acknowledgements

One would like to acknowledge my supervisor Dr Brian Rodriguez for his assistance throughout the project. Also a special thank you to Bart for giving me a thorough understanding of cell culturing and related techniques.

Thanks also to Dr Emmanuel Reynaud for his fantastic help with making the entire project very understandable and accurately predicting the outcome of the experiments. Also the project would not have been possible without the use of his optics lab and the 3d printer which was a central piece of equipment in this project.

Thank you also Dimitri for his assistance with the stereo microscope in the Conway building which was a challenge to operate at the best of times.

Also I would like to thank you to Tracy for “lending” me her pbs and to the staff at starbucks for the coffee. My only complaint with regards to the “latte(r)” was that it wasn't free...


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3.0 Abstract

Osteoblasts grown from the cell line M3TC3 were grown on synthetic polymer scaffolds fabricated on two different 3d printers. The scaffolds were produced using low cost methods and variations and attempted improvements of the manufacturing methods were utilized. Different scaffold designs were carried out ranging from fabricated lined fibril like structures to sanded structures. Fluorescent microscopy was used to identify what scaffolds showed the greatest Osteoblast growth, using the dye PI (Propidium iodide). Tensile tests of the PLA (polylactic acid) and abs (Acrylonitrile butadiene styrene) were also investigated to determine some of the mechanical properties of the materials. Human metacarpal & phalange index finger bone’s were fabricated for batch” D” and these scaffolds showed the greatest cell counts after 14 days in comparison to sanded “C” batch PLA samples.

While PLA is an excellent scaffold material, it is not suitable on its own for bone regeneration for rather as a composite for bone implants.

However it was observed that certain sanding patterns showed greater cell counts after 14 days on PLA, it is expected that combining sanding with the structures from batch “D” would further increase cell count.


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4.0 Introduction

A number of Polylactide PLA 3d scaffolds were printed from a commercial 3d printer (BFB300) and mouse osteoblasts from the cell line M3TC3 were seeded onto the scaffolds.. Certain Scaffold patterns produced much greater adhesion and thus a greater cell count. Bone Mineral density (bmd) [47] is an established useful indication of bone quality ,however structure rather, [57] is a more important factor in implantable scaffolds and indeed the arrangement of these structures can optimise factors in relation to the application of the scaffold. [14].

Currently much work is being done with regards to regenerative medicine and tissue engineering which are one and the same thing, the difference being the former is carried out in vitro and the latter in vivo. However the race is on to develop structures with better vascularisation which will lead to replacement tissue such as liver & kidneys. And Indeed for use in drug test assays. [66].

Much work has already been based on the material PLA (polylactic acid) which has been considered an extremely useful early material in implants, however its issues include, in vivo degradation’s that can trigger immune system responses from the lactic acid as well as its low modulus, however these both can be overcome by adding components such as HA (hyaluronic acid )[53] as well as additional composite materials.

In this particular project, investigation of osteoblast adhesion to PLA samples as well as materials such as PDMS & ABS samples were investigated to determine what materials and what structures optimised growth (cell count) and indeed cell density.

Adhesion forces are important considerations with regards to 3d scaffolds as if the adhesion forces are too great, the cell growth will be slower whereas if the adhesion forces are too weak the cells will not attach and therefore no growth can be achieved.

Currently much work is being done through the development of a new machine which is based on a 3d printer. Known as a bioprinter, this machine can print amarose hydrogels with stem cells or other cell lines together to form soft tissue in vitro without the use of a “hard” scaffold such as PLA or composites.

Currently one has developed such a machine in conjunction with this project but as of yet, it has to be tested with cell lines.


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4.1 What are osteoblasts?

Osteoblasts are one of the three types of known bone cell, which are known as osteoblasts, osteocytes and osteoclasts.

Osteoblasts are the migratory cells that initially propagate around a structure. Once attached and properly stimulated, they begin to form osteocytes provided there are suitable structures suitable (porous gaps). Osteoblasts are pivotal in the remodelling of bone and also in diseases such as osteoporosis (an imbalance between creation of bone and the removal of bone). Osteocytes replace bone in a process known as osteocytic osteolysis.

Osteoclasts come directly from mesenchymal stem cells in the bone marrow and considered a progenitor cell. They absorb bone while osteoblasts create bone.

4.2 Their interactions with respect to bone remodelling

Bone remodelling is partially triggered by a need for calcium in the extracellular fluid but it also occurs in response to mechanical stresses on the bone tissue.[45] This bone remodelling is achieved through the use of 3 types of bone cell that have been mentioned previously.

Communications between the cells

Cytokines act as communication molecules between cells and are part of extracellular signalling network that controls every function. This is an important factor in tissue engineering, although cell types may have common ancestry, they often have very different functions which depend on homeostasis (balance) in the body.[49].

4.3 Stem cells

Figure 4.3 mesenchymal stem cell

Stem cells can self-replicate and are capable of pro-ducing specialised cells through a process known as differentiation; they are also referred to as somatic cells [55 & 44].

These cells can be further separated into different classes known as totipotent cells that can create an entire organism. pluripotent cells that can give rise to any of an organism’s cells, but cannot form a pla-centa to grow in utero (womb). Multipotent can dif-ferentiate in to any cell line.

Some cells of blastocyst cells can form any pluripotent stem cell and can give rise to over two hundred types of cell types.

Figure 4.1 - bone cells [14] showing osteoblast, osteocytes &osteoclast


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Figure 4.31 – overview of stem cell types

Currently, there are a number of sources of stem cells divided into two main groups (multipotent and pluri-potent0. Embryonic stem cells are gathered from the inner cell mass of the blastocyst days after fertiliza-tion. Fetal stem cells are taken from the germline tissues from aborted foetuses. Umbilical cord stem cells - Umbilical cord blood contains stem cells similar to those found in bonemarrow.

4.4 Collagen

Normal bone development occurs when collagen is changed through a process known as ossification but before this, stem cells differentiate to form chondrocytes which in turn create a flexible cartilage model of the overall bone. These in turn are then converted to actual bone through a process known as ossification leaving cartilage at the ends of the bone to allow connectivity and indeed flexibility between the bones.

Chondrocytes are also known as cartilage cells, and both these and osteoblasts are formed from mesen-chymal stem cells. High density chondrocytes or their precursors are critical for cartilage regeneration, whereas bone regeneration can be based on the use of scaffolds alone.

[47].BMD (or bone mineral density) is a standard used widely in medical practice to assess bone quality, that is its stiffness and compressive strength (bulk modulus) [57] however it is not considered a good measure and is not directly related to structure [58].

Pluripotent

can generate an entire organism

Multipotent

can generate into different

cell lines

can generate more than

200 cell types

Totipotent


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Figure 4.2a – printed flexible material Figure 4.2b - Medical Stent

. Collagen is naturally degraded by a process known as metalloproteases, specifically collagenase, (both of which are enzymes) and serine proteases, allowing for its degradation to be locally controlled by cells present in the engineered tissue [35,52]

The basic structure of all collagen is composed of three polypeptide chains, which form a three-stranded rope structure. The strands are held together by both hydrogen and covalent bonds. Collagen strands can self- aggregate to form stable fibres. They can be further enhanced through crosslinking [54]. and by mixing with other polymers (i.e.HA, PLA, poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and chitosan.

4.5 Cartilage & bone formation

Cartilage is a bio macromolecular fibre composite material located at the ends of long bones that enables proper joint lubrication, articulation, and loading. This is a left over material formed from ossification. During joint motion, cartilage sustains a complex combination of compressive, shear, and tensile stresses up to 20 MPa [54] and can withstand compressive strains of 10–40%, there are three types of cartilage characterized by the composition of the intercellular matrix known as hyaline, elastic and fibro cartilage.

Cartilage also exhibits excellent lubrication properties and wear resistance. [61]. Cartilage is a specialized form of connective tissue containing the progenitor, chondrocytes cells which secrete, and are surrounded by, an extensive intercellular matrix. The strength and durability of cartilage are the properties of the matrix, which is an interlaced network of elastic fibres in collagen type II.

.Fig 4.5. image showing cartilage fibrils


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4.6 Tissue engineering

The method used in this project was in vitro tissue engineering. The seeded scaffold is created outside the host body and then placed into the body at a later stage where blood vessels are formed and attach to the new implant. Integrating the new scaffolds and cells into the host’s body. and depending on the material and its properties, that material will dissolve in vivo leaving behind a reconstructed mimic of the original tissue.

Matrices generated by 3D printing can be used for bone tissue engineering using patient’s cells seeded onto the scaffolds. The scaffolds serve as three-dimensional templates for initial cell attachment and subsequent tissue formation.

[53] In similar experiments, design of the scaffolds was done in such a way that maximum adhesion can be achieved along with rapid growth and space within the scaffold to allow diffusion of cells throughout while allowing the cells access to the medium, additionally good surface area allows the rapid propagation and proliferation of osteoblasts.

The mechanical properties of scaffold composite structures are strongly influenced by the shape, size and arrangement of the structure therefore having knowledge of these factors in both normal and osteoporotic bone is crucial in the accurate predictions of bone’s mechanical properties.

4.7 Cell adhesion forces

Due to time constrains, no measurements could be taken of osteoblast adhesion on the various materials however from other papers comparisons can be drawn;

Typical values for mammalian osteoblast cells [64,22,69] – 0.2kpa to 20kpa Measured Mouse Osteoblast detachment force on glass [23] – 1.47-1.55 microns.

Homeostasis throughout a body can only be achieved when cells attach and detach correctly. The integrity of the structure is altered according to the attachment forces that the cells have to the surface of the Extracellular matrix (scaffold) which is also known as the ECM.

Bonding to the surface is achieved through adhesion molecule classes such as integrins, caherins and selectins and there are a multitude of different adhesion molecules used to attach to different surfaces including cells.

Many diseases are in fact heavily influenced by the adhesion of key ligands or receptors in vivo. One can also determine the vitality of a cell through its adhesive ability.[51]

4.8 Creation of scaffolds – established methods There are a number of methods used for the regeneration of bone, namely;

1. Autografts that are the taking of existing bone tissue from a patient.

2. Allografts are decellurized bone taken from another person, related or not, living or dead.

3. Xenografts are materials taking from animals

4. Artificial synthetics such as polymers, metals or composites .


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Figure 4.8 Athymic mouse lacking a thymus gland with implanted human cartilage.

Other materials such as Bovine dermal collagen has been used as a suitable substrate for various 3d cell cultures with regards to tissue engineering. Honey-comb structures have a high mechanical stability under compressive loads [52] as well as physical and chemical conditions and for the exchange of nutri-ents and waste products between the honeycomb membranes, and for its ability to retain its unique structure. These prepared structures can be placed in living bioreactors such as that in Fig 4.8.

The material and design of the scaffold can also affect how the cells develop. That is the scaffold can tell the cells what function to perform. What is vital is the production of a vascular network inside of the scaf-fold that would allow it to integrate with existing tissue and restore function lost after injury or disease. [64], this is currently an area of great research. It can be achieved through a variety of methods including through co-culture with endothelial cells or by adding “signals” to promote angiogenesis into the scaffolds. These “signals” can be simply the structure of the scaffold itself and applied growth factors [24].

5.0 Scaffold Materials

Currently bone synthetic and natural inorganic ceramic materials (e.g. hydroxyapatite and tricalcium phosphate) [15 & 16] as candidate scaffold material have been aimed mostly at bone tissue engineering This is because these ceramics mimic the natural inorganic component of bone and have osteoconductive (any scaffold to which osteoblasts can attach and start bone growth ) properties However, these ceramics are inherently brittle and cannot match the mechanical properties of bone. It should be mentioned that bone is a composite comprising a polymer matrix reinforced with ceramic particles. The polymer is the protein collagen, 30% dry weight, and hydroxyapatite (HA), 70% dry weight. However, ceramic scaffolds cannot be expected to be appropriate for the growth of soft tissues (e.g. heart muscle tissue) considering that these tissues possess different cellular receptors and mechanical property requirements which is where synthetics such as PLA come in.

5.1 Natural polymers

Naturally derived protein, carbohydrate or sugar* (which is also a polymer not a monomer) polymers have been used as scaffolds for the growth of several tissue types. Collagen is however the most favourable. Collagen scaffolds can be derived from cadaver’s in a process known as decellularization after the original host cells have been flushed out and repopulating that collagen structure with cells from the host body.

Common scaffold materials include poly(lactide-co-glycolide) (PLG). PLG are hydrolytically degradable polymers that are FDA approved for use in the body and mechanically strong.

5.2 Hydrogels

Hydrogel scaffolds are appealing for cell delivery and tissue development because they are highly hydrated three-dimensional networks of polymers that provide a place for cells to adhere, proliferate, and differentiate.

They can also provide chemical signals to the cells through the incorporation of growth factors and mechanical signals by manipulation of the mechanical properties of the material. Currently, hydrogel scaffolds are being used in an attempt to engineer a wide range of tissues, including cartilage, bone, muscle,


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fat, liver, and neurons. Hydrogels have a similar macromolecular structure to cartilage, which is a highly hydrated tissue composed of chondrocytes embedded in type II collagen. Thus, cartilage is an obvious tissue to engineer using hydrogel scaffolds. To date, numerous hydrogel scaffolds embedded with chondrocytes have been synthesized and tested both in vitro and in vivo.

Suspensions inside hydrogel’s composed of extracellular matrix (ECM) proteins. As the concentration of oxygen inside 3D culture changes on the length scale of a few hundreds of microns, 3D cell-based assays should have the capability to analyze cells inside the tissue with spatial resolution of at least 100 microns.[61].

Hydrogels are widely used as in vitro ECM models to study tumour cell behaviour in three-dimensional microenvironments because they possess biophysical and biochemical properties that can be tuned to match those of the interstitial tissues into which tumour cells invade. Recently, there has been interest in determining how ECM properties such as stiffness, fibre alignment, and porosity regulate cell phenotype can influence invasive tumour behaviour. Notably, preliminary studies with metastatic breast cancer cells show that tumour cell behaviours including morphology and migration can be regulated in a strictly micro architecture-dependent manner or that is scaffolds.[68]

PLA (polylactide) polymer 4032D, a Nature Works LLC product, is a biodegradable synthetic polymer that is FDA approved and also EFSA approved (European food safety authority) it is a main component used in scaffolds and is often used in conjunction with collagen type I.

The chief advantage to PLA is that lactic acid is common in nature, and a large number of organisms metabolize lactic acid. At a minimum, fungi and bacteria are involved in PLA degradation. The end result of the process is carbon dioxide, water and also humus, a soil nutrient. This degradation process is temperature and humidity dependent.[19], however in vivo, its degradation can lead to immune system responses.

Surface qualities of the materials

.The comparison of the behaviour of deferent cell types on materials shows that they react divergently according to surface roughness. Sources have shown through Sem analysis that cell propagation is faster on smooth surfaces as opposed to rough ones where adhesion is greater.[14]

The organization of surface roughness is an important parameter to consider. In vitro, many authors have demonstrated the contact guidance phenomenon using osteoblasts or epithelial cells. Epithelial cells were markedly oriented along the long axis of 10 m deep grooves on a titanium-coated implant [51]. Adhesion is also affected by mechanical stimulation.

5.3 designing the scaffolds through software

The scaffolds are generated through software such as Google sketch, Autodesk inventor and other cad packages through a series of file conversions and processes, the finished scaffold (stl file) is loaded through the axon software from bits from bytes and then this file is converted to gcode (a form of text code providing co ordinates) that the machine can understand.

The machine firstly heats up the extruders that it will use, the temperature depending on the material to be extruded and starts creating a raft that will allow the object to sit securely to the build platform without moving. On taller objects, this raft is in fact critical as the higher the object, the more it begins to act like a lever and thus every oscillation can cause the object to dislodge and disrupt the build process..

The machine that was used to print the scaffolds for the series of experiments mentioned in this thesis is a “bfb3000” from bits from bytes, a 3d printer corporation subsidiary company. This machine uses the heated

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extrusion method. With the pla operating at a temperature of 195 celcius (568K), and the abs operating at a

temperature of 225 celcius or (598K).

5.4 How the 3d printer operates

A 3d printer is similar to some aspects to a microscope, in that it has an x,y and z stage. All of these stages move separately or together, to position the extruder at a fixed distance from the surface to which it extrudes a polymer. The extruder is heated to a temperature that makes the polymer less viscous and a stepper motor drives the semi viscous polymer out of the extruder nozzle to the surface. Correct alignment is essential, as too close, the nozzle will jam and too far, the polymer will not attach (and then subsequently jam).

6.0 Material and methods

Methods:

6.1 Batch preparation

A control batch of 6 samples was made to trial imaging and preparation. These samples were marked as “S1- 6”. Two of these samples became contaminated and were not imaged.

A further batch of 48 pdms, (Polydimethylsiloxane) samples was prepared. 24 were placed in a single array. However these samples would not fit under the stereo microscope and were abandoned. However, strong growth was observed on these scaffolds through the use of an optical microscope.

Another 24 samples were separated into 4 trays of 6. Four of 6 samples were from the same sample type, the fifth being an empty control section with the 6th spacing holding a random floating sample.

6.2 Preparation of cells

After a set period of time the cells in the culture container were removed from the incubator and observed under a standard farfield microscope.

To determine the number of cells, a sample of cells can be extracted and placed in a measuring device. The cells are drawn across the slide by capillary forces and the slide is then placed under the microscope. Taking three random squares and using a counting device, three counts were carried out on the number of observed cells. This count was divided into three to give an average and this average is the number of cells per 0.1micro litres. (ul) The number of cells required for each well was 100,000.

6.3 Preparation of cells for seeding onto platforms

Medium was removed from fridge and heated in bath at 37 degrees celcius. (310K) – trypsin was also removed from the fridge and heated in the bath at 37 degrees for 15 minutes. The medium was extracted from the cell culture container either by pouring into a waste container or through the use of a micropipette. Trypsin was added equally across the container, after which all liquids were removed with a micropipette and placed in a separate container..

The extracted trypsin and cells were separated with in a centrifuge at 2000rpm for 5 minutes.

After removing the trypsin, fresh pbs was added and the cells diffused trough the medium with the aid of a micropipette. The cells were then extracted and seeded onto the scaffolds.

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Investigation of Osteoblast Adhesion on 3d printed scaffolds – Jemma Redmond –– 09260340 – Msc

Nanobioscience 6.4 Scaffold Sample Preparation

Fig 6.4 – Cleaning of samples

The samples were separated into separate containers, then cleaned with pbs and placed in the ultrasonic bath for 30 minutes. The samples were then autoclaved to remove any bacterial contamination for twenty minutes

Each sample was placed in a well and held in place (where required) with a micropipette as it was discovered that the samples tended to float. Each container was marked with a batch number and dated. A medium was prepared and heated in a bath at 37 degrees for 20 minutes. Prepared cells from cell line MT3C3 were seeded onto each well. The medium was added to each well submerging the entire scaffold and micropipette (where possible). The samples were then placed back in the incubator set at 37 degrees or 310K.

6.5 Sample preparation for imaging

One batch was removed after 24 hours, 3 days, 7 days and 14 days. The medium was removed from each well and pbs was added. Pbs was placed in a heated bath at 37 degrees for 20 minutes. The medium in each well was extracted and pbs added to each well matching the level of medium extracted.

PI ( a fluorescent marker) was added to each well and then placed in the incubator for 10 minutes.

After 10 minutes, the pbs / pi was removed and fresh pbs added, the samples tray was wrapped in aluminium foil to reduce the chances of bleaching prior to the samples being placed under the microscope. The samples were imaged under a stereo microscope with 5 areas being captured on camera. 2 images were taken – one at 8x magnification under normal light conditions and the other at 45x magnification under fluorescent conditions, where it was deemed necessary, further images were taken. The last 2 images were taken of the well itself.

6.6 Design of the “S” samples

The S samples were the precursors to the B samples and thus the same structure types were present bin both of these sample batches.

6.7 Design of the “A” samples

The “A” samples consisted of PDMS moulds. PDMS, known as Polydimethylsiloxane is an inert silicon synthetic scaffold. The moulds were 50 microns across and manufactured to originally monitor the growth of fungal spores. The moulds were kindly supplied by Dr Emmanuel Reynaud.

These scaffolds where easy to view under a farfield microscope however they proved to be difficult to see when immersed in pbs. In addition, alcohol and the autoclaving process appeared to damage them. These scaffolds can be viewed under Appendix’s A,B,C,D & S.

6.8 Design of the “B” samples

A number of designs were chosen and created on google sketch. Structure types of grooves, microtubules

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Investigation of Osteoblast Adhesion on 3d printed scaffolds – Jemma Redmond – – 09260340 – Msc

Nanobioscience and circular structures amongst other were generated.

6.9 The structure types that were used for the scaffolds.

Design of the “C” samples

The c samples were created as uniform slabs that could fit into each well of the 6 well sample plates. C2 to C4 were sanded with C1 being unsanded. C5 and C6 being the control and float respectively.

Non sanded C1 C7 C13 C19 Sanded left C2 C8 C14 C20 Sanded up C3 C9 C15 C21 Sanded left and up C4 C10 C16 C22 Control C5 C11 C17 C23 Floating Sample C6 C12 C18 C24

Table 2. “C” samples – Sanded and non sanded pla samples

6.91 Design of the D samples


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From these set of samples facsimiles of actual metacarpals and phalanges were created with a modified structure that would allow the capture of cells along with smooth ridges to allow the cells to propagate quickly. While the structures were porous it was not possible at this time to create a more porous structure and the purpose was merely to test the adhesion of Osteoblast cells to the various structure types.

As incubator size and dish size were limiting factors this limited the growth of cells as it was not possible to completely immerse the metacarpals without possible spillage and contamination of the other well plates.

Metacarpals D1 (24 hours) D6 (3 days) D12 (7 days) D18 (14 days)

Figure 6.91a Human Hand Anatomy showing metacarpals & phalanges used in sample batch “D”


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Proximal phalanges

D2 (24 hours) D7 (3 days) D13 (7 days) D19 (14 days)

Middle phalanges D3 (24 hours) D8 (3 days) D14 (7 days) D20 (14 days) Distal phalanges D4 (24 hours) D9 (3 days) D15 (7 days) D21(14 days) Control D5 (24 hours) D10 (3 days) D16 (7 days) D22 (14 days) Floating Sample D6 (24 hours) D11 (3 days) D17 (7 days) D23 (14 days)

Table 6.91b. D samples preparation

All of the samples above were harvested after 24 hours, 3 days, 7 days and 14 days.

The files for the metacarpals and phalanges were taken from thingiverse http://www.thingiverse.com/thing:15342 [26] which is opensource and permitted to use for non commercial applications. These files were converted to gcode for operation with the printer. They were however modified (laid flat) to enable better printing and alter the structure.

For the D set of samples a set of 4 of each bones were created (from the left index finger) and then subsequently replicated 4 times (making a total of 16 samples). In one set of samples, the bones were printed upright rather than flat., the remaining two spaces on each plate well contained the control (no scaffold, just cells) and the floating sample.

But it was discovered that, after several failed attempts at printing these, the bones printed every time correctly when they were printed flat and with support structures to ensure that they were printed correctly. One way of course to overcome this was to embed the bone in a support structure but this would require more time, affect the surface topology (and increase defects) and require more energy and materials.

In addition it was observed from previous samples manufactured in this manner, that the samples that were printed upwards or vertically tended to be weaker than those that were printed flat. This is because the printer prints in layers and those layers are shorter and weaker when printing vertically upwards and larger and stronger when printing horizontally.

It was also observed that the osteoblasts were more numerous on the horizontally printed samples. But this is possibly because the ridges or ledges are horizontal thus trapping the osteoblasts and preventing them from falling off the scaffold to the bottom of the well.

However, from subsequent observations from random samples, it was observed that the cells did indeed stick to the underside of the PLA samples no matter their shape or configuration. Although this was not to the same degree as those that were on top.

6.92 Observed Osteoblast adhesion

The sample area consisted of ridges formed by microtubule like structures that were extruded from the nozzle of the 3d printer. The cells did not necessarily congregate in between the ridges but rather grow on the ridges themselves, forming straight lines. These straight line patterns were observed on the surface of bottom of the well.

7.0 3d printers

The extrusion 3d printer and its operation

http://www.thingiverse.com/thing:15342


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These machines are excellent for rapid prototyping as opposed to manufacturing. Therefore this makes them an invaluable tool in tissue engineering. One can create multiple structures on different materials through open source software and produce those structures in minutes.

The machine itself operates in a similar fashion to most far field microscopes except that there are automated motors that can control a heated extruder head to position plastics in a precise location. A variation of this machine is the bioprinter that can extrude cells directly into a hydro gel such as amarose.

7.1 Software

Software that was used to design the scaffolds on the bfb3000

Google sketch 8

mesh

Autodesk inventor

axon 1

axon 2

Axon version’s 1 and 2 – this was the software that directly controlled the bfb 3000 and allowed limited modifications of the scaffolds. It was determined from this software that hollow sphere like internal structures were the fastest and lightest to produce. Replicator g – this opensource software was used to centre the objects created or downloaded from google sketch – the axon software had a limitation where it was unable to centre the scaffold onto the build platform. Google Sketch version 8 – this freeware program allowed one to create objects and export (with the use of an stl plugin) an object to the axon software suites. Mesh – mesh was able to change the file extension of the google sketch 3dp files to stl files that could be used on axon Slicer – Can take mri and cat scan images and print the objects as an stl file or a volume file. ImageJ – A cell counter program that works best when the images from the microscope are clear – unfortunately in my case – this was not so and a manual count had to be carried out. Interface software for the zeiss v12 – proprietary software that operates with the microscope to output jpeg files.

7.2 – The bioprinter – a “phenotype” of the 3d printer

The bioprinter is a variation on the 3d printer. It is also quite similar in operation to most microscopes or the atomic force microscope in that it uses 3 motors to control the direction of movement of the printer head. This is achieved manually or through a pre programmed set of co ordinates that are stored in a text file variation known as gcode.

The extruder head in this case is different to a standard 3d printer in that the head consists of two syringes that contain separate amarose hydrogel that is a viscous liquid that is rich in nutrients. While the other syringe contains the cell line or stem lines. The two syringes are in turn wired to a compressor at 80 psi which is controlled via two control lines from a mainboard (arduino mega 2950) that uses actuators (solenoids) to turn air pressure on and off.

This pushes the cells out in clumps or spheroids in a fixed position that is only as accurate as the motors and the software driving the machine.

The cells are kept at 37 degrees by the use of a conduction plate controlled by a separate line that touches or conducts heat through aluminium foil surrounding the syringes.


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This heat not only keeps the cells warm, but it also reduces the viscosity of the hydrogel making extrusion easier. The entire machine is enclosed in a extractor hood to reduce the probability of contamination from pathogens.

In reality, the construction of such a machine is relatively simple, but getting the machine to operate correctly and reliably can be difficult.

However this piece of equipment will rapidly become the standard for future tissue engineering applications, currently there are two commercial versions of this machine on the market, one from organovo and the bioplotter from enviontec gmbh.

8.0 PLA versus abs

It has been found that abs produces an odour assumed to be toxic from the melting of the plastic and extrusion onto the surface of the platform of the bfb3000. It has also been observed rather than proven that the ABS is subject to greater thermal expansion and thus warping increases the probability of failure of the construct of whatever it is that is being made. It was observed that this is in fact less so when using the pla. ABS also tended to fail when the machine was run faster due to thermal expansion.

Using two materials at three times the normal running speed. The thermal expansion was greatly increased – this could be partly due to the rapid movement of the robotic arm causing airflow currents to be formedaround the object. On the other hand it was also assumed the time taken for the raft to cool was not great enough and this caused the ABS raft to warp damaging the PLA component that was made on top of the raft.

Also ABS rafts separated easily from PLA suggesting that more complex internal structures could be fabricated with these materials as the PLA can be dissolved through hot water.

The PLA on the other hand separated not as well on the abs raft. Again this can be assumed to be thermal expansion on the part of the ABS, causing it to expand and tighten around the PLA.

9.0 Results

The arrangement of the cell plates differed for the first 3 sets of samples and afterwards were standardised

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for simplicity with the later “C” and “D” batches.

Figure 9.1a- Force Curve for Pla

Figure 9.1b- Force Curve for ABS – Raw Abs versus raw Pla

Calculating youngs modulus for abs and pla

Exte

nsi

on

mm

0.

088

0.

18

0.27

2

0.36

4

0.45

6

0.54

8

0.64

0.

732

0.

824

0.

916

1.

008

1.

1

1.19

2 1.

284

1.

376

1.

468

1.56

1.

652

1.

744

1.

836

1.

928

2.

02

2.11

2

2.20

4

2.29

6

2.38

8

2.48

2.

572

2.

664

2.75

6 0

50

100

150

200

250

300

350

forc

e (N

)

PLA ex

t (m

m)

0.10

4

0.21

2

0.32

0.

428

0.

536

0.

644

0.

752

0.

86

0.96

8

1.07

6

1.18

4

1.29

2

1.4

1.

508

1.

616

1.

724

1.

832

1.

94

2.04

8

2.15

6

2.26

4

2.37

2

2.48

2.

588

2.

696

2.

804

2.

912

3.

02

3.12

8

3.23

6

3.34

4 0

100

200

300

400

500

600

700

800

forc

e (n

)

ABS


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The spec sheet elastic modulus for pla 3440Mpa The spec sheet elastic modulus for abs NA A typical elastic modulus for pla 350 – 2800Mpa A typical modulus for abs [68] 2.3Gpa

Fig 9.3 – Table of values for Elastic modulus

9.4 How data was gathered

Any counts that were not 45 times magnification were ignored. The fifth imaged area was also ignored as this showed cell count on the surface of the plate.

Samples S1 to S4's cell counts were disregarded as they were used for calibration and initial tests although initial results were used to create and shape the other batches of samples.

9.5 Scaffolds – which were best?

Any structure that had an interconnecting matrices or matrix that consisted of continuous microtubule like structures showed the greatest cell attachments. Any disordered states did show cell attachment however the cells did not “line up” or connect well together. If the microtubule like structure was straight or curved, they would attach.

The cells also attached rather well to the sides of the scaffolds and indeed to the underside of the scaffolds They also showed internal migration where the scaffolds were openly porous. However it was difficult to determine if the cells had indeed migrated internally throughout most of the scaffolds as imaging of the nucleus could only take place at the outside of the structure.

A way to overcome this would be to slice the scaffold into sections and image them that way. However it was noted initially that it was quite difficult to cut the scaffolds whether they be “PLA” or “PDMS” and thus to do that and expect to image living cells would be very difficult indeed.

Anything with microtubule like structures showed the greatest concentrations. Damaged areas of scaffolds also showed strong adhesion due to roughness or irregularity of the surface, however looking closely at the damaged areas showed internal structures of the printed materials that resembled the wave like structures one would fine in regular human tissues or commercial stents. There are no clear images of this as only a handful of these damaged scaffolds appeared and were also quite difficult to image due to problems operating the v12 microscope


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Figure 9.5a samples over 14 days

Figure 9.5b – C sanded samples over 14 days

Figure 9.5c– d metacarpals & phalanges over 14 days

Tubles/fibrils Circular Grooves spheres Control Floating

0

10

20

30

40

50

60

70

Sample type

Ce

ll C

ou

nt

(N)

"B" samples over 14 days

24 hours

3 days

7 days

14 days

Not Sanded Sanded Left SandedUp Sanded left and up

Control Floating Sample

0

10

20

30

40

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Sample type

Ce

ll C

ou

nt

"C" Sanded Samples over 14 days

24hour

3 days

7 days

14 days

Metacarpal Proximal phalange

Middle phalange

Distal Phalange

Control Floating

0

20

40

60

80

100

Sample types

Ce

ll C

ou

nt

(N)

"D" Metacarpals & Phalanges over 14 days

24 hour

3 days

7 days

14 days


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Taking the best sample from each batch and doing a 14 day comparison shows the mimic of the metacarpal structure to perform the best amongst the samples, despite the micropipette covering about 25% of its surface and despite the fact that there was 20% less cells seeded onto these scaffolds and in addition, the metacarpals were not fully immersed in medium throughout as there was insufficient medium to cover them. The proximal phalange was fully immersed in medium throughout.

The undersides of the samples were randomly checked and did show Osteoblast adhesion on the underside and along the sides, this was also the case with the metacarpal mimics however despite there being less surface area above the sample (immersed) there was in fact a greater number of cells here.

Figure 9.5d – a comparison between batches

9.6 Images and cell count

The zeiss V12 stereo microscope that was used had some “technical” issues. That is the alignment of the camera was found to be out of alignment with the viewing eyepiece, thus what was physically observed differed significantly from the images that were taken from the camera.

The images that were taken from the camera tended to be cloudy and difficult to align meaning that the software package “imagej” was unable to do a direct count on the number of cells observed thus a manual count was required in more than 500 image although in batches “C” and “D”

If the images were ambiguous or the cells appeared detached, these were disregarded in all counts thus ensuring a conservative counting method.

In earlier versions of the sample imaging, an 8 times magnification was initially taken and then a larger magnification was taken. It was determined shortly after this that in order to make things consistent, one had to have two standard magnifications, thus 8x and 45x were chosen. All cells counted were at 45x and any counts outside (with the exception of S2) were ignored or regarded as “0”.

As “imagej” would not count the cells from the images obtained, a manual count was carried out on all 1300 images. Any images that were indistinct were disregarded, any cells that appeared to be floating were ignored. So the numbers obtained from the photos were highly conservative and further more the camera (which was not correctly aligned with the viewer) showed a lot less cells that what was actually seen by eye.

S A B C D

0

10

20

30

40

50

60

70

80

Batch

Ce

ll C

ou

nt (

N)

A comparison between batches

24 hours

3 days

7 days

14 days

24


However the results obtained whilst conservative, are indeed accurate.

9.7 Magnification

It was determined from trials at batch “S” that setting the magnifications to 8x and 45x allowed a good contrast of the surface features.

10 images of each sample was carried out with 5 being of 8x magnification and 5 being of 45x magnification, however where it was deemed necessary, further images were taken, but only one image of 45 times magnification was utilised ( and that showing the lower cell count). Cell counts on the bottom of the well plate were disregarded for the purposes of this project but those in the control sample were counted.

10.0 Discussion

10.1 Improvements made to the scaffolds

From earlier versions of the “S” samples, it was observed that microtubule like structures that created groove effects around their sides showed the greatest cell attachments but not necessarily the greatest cell adhesion Thus in later versions, “B” samples, this was again observed, thus, these tubules were selected and improved upon in batches “C” and “D”. In batch “D”, the metacarpals showed the greatest cell counts and were largely consisting of these structures.

10.2 Improvements made to the 3d printer

Upgrading of the axon software to version 2 allowed the resolution to be taken down to 125 microns providing greater detail and features in the scaffolds.

In addition there were 4 types of infill pattern that could be applied. Testing out all 4 infill types versus speed showed one type to use less material, build faster, use less energy and be lighter. This happened to be the same infill pattern as you would find on a standard lego abs brick.

The machines speed could also be increased by 40% but only using PLA as the material. The abs material tends to warp due to thermal expansion and is very dependent on room temperature. It was found that the warping is also proportional to the operating speed of the machine.

PLA is the only material on a standard 3d printer that was observed to operate at increased speeds with reduced failure rates. This reduces energy costs, materials and build time which means that products / implants can be produced quicker and cheaper.

Running the cables outside the machine

One problem with printing with the bfb3000 was that the material was that the roles are placed under the machine and fed through to two inner tubes which in themselves are very difficult to load. The material also had a tendency to break inside the tubes and this made it very difficult to remove the material from the tubes, From taking what was learnt from microbending in optics, (where that the angle of the fibre optic strand is too great., the material can in fact deform and break) and applying this to the problem with the cable breakages, it was found that repositioning the cable reels at the top of the machine and fed directly into the extruder reducing the number of turns through which the cable was fed, this greatly reduced loading times and lengthened operating times.


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Running the machine at high speeds

This can reduce material and energy costs as well as time costs. However it has been observed that the overall quality of the end product is reduced that is the finish does not appear to be as good as printing at a slower speed. But the overall functionality of the object appears to be the same. However surface roughness is also greater in objects printed faster.

10.3 How could the process be improved?

If the scaffolds have more interconnections, the greater the mechanical strength and perhaps the better the propagation of the cells throughout the structure however there would be a trade off between flexibility and mechanical stiffness

10.4 Straight Lines / Bridges

Where the growth on the manufactured surfaces was greater where the scaffolds that had straight lines or grooves. These would appear to resemble microtubules or fibrils and the osteoblasts lined up very well on these structures.

10.5 Floating Samples

The floating samples were random scaffolds but not held in place with a micropipette tip. These showed the most cells on the surface as opposed to the other structures but this is possibly largely due to micropipette covering a large part of the surface. Thus a greater cross sectional area was available.

The cells also lined up on these samples but not as well as those that were embedded in the medium. Despite the limitations of the microscope, the data obtained whilst flawed matches results from other papers and what was expected. [2&3]

10.6 What samples were the most effective?

PLA sample 3 (B9, B10, B11, B12) showed approximately double the number of cells on the scaffold as opposed to the control. This structure had a square grid like structure overlaid with microtubule style bridges at a 45 degree angle. Growth was strongest on the microtubule like structures, particularly where there were 3 points (x,y,z) of contact.

PLA Sample 1 (B1, B2, B3, B4) also showed similar growth however was quite similar to sample 3 with the spacing being closer together.

PLA Sample Batches D showed the greatest number of cells on the surface and in alignment in comparison to the other samples. Interestingly, these samples had less cells, less surface area and were not completely immersed in medium.

The “C” batches of samples have been prepared with sandpaper. Various groves have been created on these scaffolds. In addition the “phenotype” of these scaffolds would be simply a variation of lines and spacing, previous papers have shown these methods to be effective with titanium but depending on how the roughened surface was applied.[1,2 & 3]

From earlier observations, it seemed logical to assume that the lined structures would produce better results than the next two batches of samples which also have these features. They are labelled as Batch “C” and batch “D”. Batch D are stl copies of human metacarpals & phalanges with the lines arranged horizontally.


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10.7 Limitations of the microscope

Due to poor imaging from the camera, many images produced were not useful and reflected badly what was seen by eye. In addition, due to the poor quality of the images, the image processing software used (imagej) was unable to count any cells in the picture and thus a manual count was carried out on each photo.

The samples also had a low level luminescence emanating from them, this was overcome by using flurophores activated by an appropriate wavelength that is 488nm excitation and 543nm emission

It was not possible to see the cells through an optical microscope without fluorescence as the background (certain scaffolds were white, however black also showed fluorescence but to a lesser degree) however it was possible with pdms scaffolds to visually see the scaffolds and cells.

10.8 Printing of scaffolds through low cost 3d printing

A portion of the initial samples were created on a makerbot thingomatic 3d printer – however due to errors in sample preparation, these samples were abandoned. It was decided it would be more prudent to use one machine to produce all of the samples (which was the bfb3000). The thingomatic can be constructed for under 1500 Euros at the time of writing and it is indeed feasible to manufacture implantable scaffolds with this machine.

The next step up in cost is the bfb3000 and was the main workhorse for the manufacture of the scaffolds. It has a capability of printing from 125 to 500 microns. From paper [35]. it has been suggested that 100 to 200 micron spacing in scaffolds allows for optimal permeation of the scaffold by the osteoblasts.

However data shows that the cells do not necessarily tend to grow in grooves but rather loosely follow them. It would appear that the porosity [35] of the material may be more important and that can be “tuned” through the “infill” settings on the software (skeinforge).

That is the density of the structure can be controlled using the infill feature and thus the strength of the material can be increased. However paper [45] suggests that density (BMD- bone material density) while it does have an “impact” on strength it is not an accurate indicator but rather the structural arrangement of the scaffold or material is far more important. Indeed structure seems to be the key to the usefulness of an implantable scaffold.

10.9 The materials used in the creation of the scaffolds

PDMS – was manufactured through moulds, the cells did indeed stick well to these but the structures were not helpful in the growth of the cells. That is the cells built up in the pores and then connected together. Where the scaffold was cut the cells grew rapidly along the edges connecting the separate sections together.

ABS– ABS is known for its toxicity and requires a higher temperature to run. Its advantages tend to be lower costs and increased strength, however it was found that the abs had a high tendency to warp possibly causing the overall “build” (the assembly of a product) to fail. This became more evident when the material was printed at higher speeds or the room temperature had changed.

PLA on the other hand showed good strength and little evidence of warping. Thus one was able to operate through the 3d printer at higher speeds with no significantly increased probability of failure.

While more expensive to purchase, it does in fact use less energy as it operates at a temperature of 468K.


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The issue with both materials is of course strength or mechanical stiffness but depending on the application, for example soft tissue implants, this may not be an issue.

10.10 The problems encountered in the creation of the scaffolds

Warping of the ABS – Due to thermal expansion issues, the ABS has a tendency to curl on the platform distorting the build and causing it to fail. If the ambient temperature of the room is too high then this makes the problem worse. If the temperature is too low this can also have a negative effect. The problem is also exacerbated through fast builds.

Incorrect alignment of the extruder – If the extruder head hits the platform, this can cause the head to bend and thus jam the extruded materials. If the head is too close to the bed, the nozzle may hit and cause the extruded material to jam, and if the head is too far away from the build platform, then the extruded material may not make contact.

Dimension issues due to software – dimensions can be measured in Google sketch however when the build file (3ds) is transferred to other software packages for processing, the dimensions are lost and thus one needs to rescale the scaffold each time.

Jamming of the extruder head – this can because of a bad filament, a break in the filament, incorrect alignment of the extruder or damage to the nozzle.

Incorrect alignment of the print platform – mechanical stresses cause by moving parts can lead to the platform going out of alignment resulting in a failed build.

Breaking of the material reel – this issue was resolved by removing the reels from the underside of the bfb3000 and creating a holder suspended from a shelf above the machine, placing the reels in this and directly feeding the reels into the extruders.

10.11 The solutions developed in the creation of the scaffolds

Printing materials flat on the surface of the platform with a support structure (particularly bone) produced stronger bones and less defects. If the bones are printed vertically, the further up that the head prints, the greater the movement of the build structure. This increases the probability of surface defects or indeed complete failure of the build.

However printing horizontally overcomes this issue. In addition the alignment of the groves mimics more naturally the arrangement of the collagen fibrils in bones.

10.12 On growing the cell culture on the scaffolds

All cells were directly applied to the surface of the scaffolds in one area although the cells migrated throughout the scaffold and the bottom of the well plate. In Samples “S & D” there were less cells available in comparison to wells “B” & “C”, yet, images and data shows that this in fact “S” and “D” showed greater concentrations of cells. However this does not appear to have a major impact.

10.13 Imaging of the scaffolds – Problems with solutions

Properly labelling of the containers to avoid confusion (alcohol dissolves ink) and imaging without the container lid on helps improve imaging dramatically. A properly calibrated confocal microscope using fluorescent imaging would be extremely helpful in building up a good image of the 3d scaffolds giving a more accurate representation of the cell count.


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The cell count that was recorded was rather conservative as it what was imaged through the attached video camera did not match what was seen by eye.

10.14 What could be done better next time?

Positioning of the scaffolds – during preparation it was observed that the scaffolds had indeed a capacity to float in the medium thus exposing the top of the scaffold to the air.

A non toxic adhesive was used to attach the scaffolds to the bottom of the well however this proved ineffective. Therefore a mechanical approach was required and initially pieces of raw pla cut from the reel prior to extrusion. These strips of raw PLA were measured to be slightly greater than the diameter of the well plate and allowed them to hold the scaffolds securely in place at the bottom of the well. Manufactured strips were also made but these shrank too.

As autoclaving was necessary, it was found that these raw samples shrank rendering them unfit for purpose. To overcome this problem, a quick solution was needed and thus the micropipette heads were found to suit those requirements amicably.

Each micropipette head has a coating that prevents cells from sticking to them and thus they functioned rather well in keeping the scaffolds in position. However the micropipettes did reduce the surface area of the scaffolds by up to a third meaning that there was less available area for the cells to grow upon. Therefore with regards to floating samples that were submerged the surface area was much greater and this could of course allow for greater cell coverage and a greater surface count. However as these floating samples were random samples and thus random structures, one cannot make a direct comparison between the floating samples and the fixed samples; perhaps this may be something to investigate next time.

10.15 why do the cells not fit into the grooves?

Figure 10.15a – d72a

Generally, from the images, the cells do not sit in the grooves,(although they would appear to be captured by them initially) but rather on the ridges surrounding them. The cells also are found on any exposed surfaces, on sides and occasionally in between openings in the scaffolds. However they favour microtubule like structures.

However the cells appear to adhere less to flat surfaces as well as grooves but rather use the grooves as a directional guide. It is assumed that a layered series of grids or a matrix would provide optimal growth. If perhaps these surfaces were smooth, but interspaced with rough ridges then not only would the cells spread rapidly but also gain better adhesion around the rougher sections. To promote Osteocyte development, further gaps in the microtubules would allow the cells to creep in.

This is possibly due to the requirement that cells derive nutrients from the surrounding medium. By attaching to a flat surface it has a fixed area of interaction surrounding the cell. If on the other hand it is on a ridge. That area of interaction is increased. If is in a groove, that area is decreased. If it is in between two tubule like structures then not only has it anchored itself but it has also increased its area of interaction however as this surface feature is not so common, this is less likely to occur.


29

Figure 10.15b – Composite 21-22

Generally from the following images, one can see that the cells certainly attach well to any ridge or structure that it antenna like or branches out.

It was also noted that cells attached to the underside of the scaffolds as well as to the sides of the scaffolds although with regards to the former, there were fewer cells here than were observed on the top surface of the scaffolds.

It was also observed that a number of the cells migrated to the underside possibly due to mechanical vibrations from the incubator. It is also possible that cells that did not quite attach to the plate well surface moved under the scaffolds and attached there perhaps also due to the scaffold moving about slightly in the medium.

It can be seen from the tensile graphs (9.2a and 9.2b) of the two materials that neither is suitable for long term bone replacements but rather as a short term support structure. A composite material containing synthetic and organic compounds would possibly work best for long term use.

Figure 10.15c - d28b

D28b shows an ABS sample - however this is a floating sample – as can be seen from the imaging – the cells have clumped together and detached from the surface – indicating toxicity which is what was expected.

From what has been observed, one can assume from the data that grooves and lines are highly instrumental in the guidance of Osteoblast cells

Adhesion of the Osteoblast is essential in allowing it to populate the surface of a structure. The nucleus of the cell does not i fact become embedded on the surface but is anchored there by its membrane which expands across the surface seeking out other osteoblasts to create a simple network.

Adhesion is dependent on factors such as time, the surface material, points of contact that is with regards to the latter if the cell has more points of contact, naturally it should have greater adhesion with the surface that it attaches to.

PLA would appear to be more favourable than glass for cell adhesion also smoother features of the pla allow the rapid propagation of cells. This can be observed in the images below

One explanation is that the cells have a tendency to stick to the sides or near to the sides of the scaffolds, namely anywhere there is a protrusion, thus the smooth road like structures are in fact tubular It is

30


assumed that if the Osteoblast is on a protrusion it has a greater probability of interaction with nutrients

and other cells while remaining anchored.

Rough like surfaces that have been sanded so in fact show greater number of cells after 7 days although growth is slowed in these cases as can be seen from the preceding days It is assumed that the cells are able to attach better to rough surfaces and indeed have even greater adhesion in these regions. So this surface may be suitable for long term implants such as load bearing hips.

PLA is an excellent material for a cell growth however there are indications of toxicity [19] suggesting an inflammatory response is likely in vivo with degradation hence the reason why composites materials are added to negate this effect and perhaps bolster the mechanical stiffness of the scaffold.

Abs would not appear to be a suitable material due to its toxicity albeit no proper toxicology assay was carried out due to contamination of limited samples and then of course time constraints. However samples were added to C and showed the cells indeed clumping together indicating toxicity.

Abs has a higher modulus in comparison to PLA, it has a lower unit cost too however PLA is far superior material as 1 it is not subject to thermal expansion to the degree that abs is. Builds generally succeed with this material thus there is a lower probability of failure making them more cost effective in the long term for production purposes.

Additionally the material is made from biodegradable materials and operates at a lower temperature meaning that less is required in its manufacture and disposal.

Thus a composite version of PLA would indeed be highly suitable as the basis for soft tissue engineering such as skin or organs such as bladders, kidneys or livers, provided of course the correct structure can be made at the correct resolution which is quite possible with a bfb3000. Currently the machine can go to 125 microns but by changing the extruder head and reducing the aperture from 0.5mm to 0.3mm it should in fact be possible to reach beyond 125 microns.

10.15 Other uses for pla implants

Pla can be dissolved through high temperatures or perhaps vibrations, suggesting that the material could in fact be used as a drug delivery system. It is also feasible that the scaffold could be dissolved externally through non invasive methods.

Further work (and funding) is required before this can be investigated further.

10.16 Ideas and method for the creation of new scaffolds

It has been noted that the circular infill is the most energy efficient in terms of mass used and the time it takes to print an object. The internal structure fill has no significant effect on the exterior of the scaffold although interestingly one can apply a honey comb infill to the internal structure and then terminate the build before completion leaving the internal structure exposed.

By changing the resolution one can also control the spacing between these structures thus creating a scaffold structure that will allow flexibility, good cell adhesion and good porosity for cell permeation.

11.0 Summary

Approximately 100 samples were created with approximately 78 of those having been imaged. Data suggests that certain structure types have a greater concentration of cells attaching to the surface.

31


Generally cells showed greater attachment to ridges or exposed edges where there was greater probability

of extracting nutrients from the surroundings whilst providing a point of attachment.

The scaffolds that tended to show more of this were the microtubule like line structures in the “S” samples and “B” samples and again at the sides of the “C” samples and on ridges on the “D” bone samples.

With regards to the “C” samples, a standard set was created however it was found that sanding the samples while it did improve adhesion did not show a greater number of cell attachments compared to the sides of the sample where there were microtubule like ridges. It is known from other results that smooth surfaces promote fast growth of osteoblasts across the surface while the adhesive forces are known to be weaker on smoother surfaces. Whereas on rougher surfaces the slower the progress but the greater the adhesion forces. There was however microtubule like structures going across the surface of each sample but these tended to be flatter in comparison.

The “D” samples were downloaded from thingiverse and modified. These modifications changed the structure to mimic collagen fibrils as would be found in human bone; however these were on a microscale as opposed to a nanoscale.

These particular scaffolds proved rather successful despite the fact that the scaffolds were not completely immersed in medium or PBS due to sample dish limitations. In addition there were fewer cells used for the D samples yet despite these disadvantages, one can see that the cell counts are comparable in number to the other sample batches; however it is known the difference between the adhesion forces on the d samples in comparison to the c samples.

Overall there was a tendency for osteoblasts to be captured by grooves and then spread out from those grooves over smooth surfaces (bridges) and had a tendency to move to the top of the surface where possibly there was greater access to nutrients. There was evidence to show that the cells migrated internally however without slicing the scaffolds it was not possible to investigate whether this was in fact the case.


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12. 0 References

[1]. BME504 Tissue Engineering and Regenerative Medicine – Scaffolds Dr George A Burke Enhanced osteoblast adhesion on transglutaminase 2-crosslinked fibronectin J. Forsprecher Æ Z. Wang Æ V. Nelea Æ M. T. Kaartinen Biomaterials 21 (2000) 667}681 Review [2]. Osteoblast adhesion on biomaterials - K. Anselme* Institut de Recherche sur les Maladies du Squelette, Institut Calot, Rue du Dr Calot, 62600 Berck sur mer cedex, France - Received 11 January 1999; accepted 21 October 1999 [3]. Topography effects of pure titanium substrates on human - osteoblast long-term adhesion - K. Anselme , M. Bigerelle - Laboratoire de Recherche sur les Biomateriaux et les Biotechnologies, Universite du Littoral Cote d’Opale, 52 rue du Dr Calot, 62608 Berck sur mer cedex, France Institut de Chimie des Surfaces et Interfaces (ICSI), UPR CNRS 9069, 15, rue Jean Starcky, BP 2488, 68057 Mulhouse cedex, France, Laboratoire Roberval, FRE 2833, UTC/CNRS, Centre de Recherches de Royallieu, BP 20529, 60205 Compiegne, France Equipe Surfaces et Interfaces ENSAM Lille, Laboratoire de Metallurgie Physique et Genie des Materiaux—CNRS UMR 8517, 8 Boulevard Louis XIV, 59046 Lille cedex, France Received 7 June 2004; received in revised form 29 November 2004; accepted 30 November 2004 [4]. Effect of a gold–palladium coating on the long-term adhesion of human osteoblasts on biocompatible

metallic materials - Karine Anselme a,⁎, Maxence Bigerelle Institut de Chimie des Surfaces et Interfaces (ICSI), UPR CNRS 9069, 15, rue Jean Starcky, BP2488, 68057 Mulhouse Cedex, France - Laboratoire Roberval, FRE 2833, UTC/CNRS, Centre de Recherches de Royallieu, BP 20529, 60205 Compiègne, France - December 2005 [5]. Biomaterials from beer manufacture waste for bone growth scaffolds - M.A. Martin-Luengo , M. Yates , M. Ramos , E. Saez Rojo, L. Gonzalez Gil & E. Ruiz Hitzky, A.M. Martinez Serrano Institute of Materials Science of Madrid (CSIC), Calle Sor Juana Ines de la Cruz 3, Cantoblanco, 28049, Madrid, SpainInstitute of Catalysis and Petroleochemistry (CSIC), Calle Marie Curie 2, Cantoblanco, 28049, Madrid, Spain Centre of Molecular Biology Severo Ochoa (CBMSO), Calle Nicolas Cabrera 1, Cantoblanco, 28049, Madrid, Spain 09 Mar 2011 [6]. Multizone Paper Platform for 3D Cell Cultures Ratmir Derda1,2*¤, Sindy K. Y. Tang1,2., Anna Laromaine1,2., Bobak Mosadegh2., Estrella Hong1, Martin Mwangi1, Akiko Mammoto3, Donald E. Ingber2,3,4, George M. Whitesides1,2* [7]. Three-Dimensional Printing Using a Photoinitiated Polymer Joseph Muskin* and Matthew Ragusa The Center for Nanoscale Chemical-Electrical-Mechanical Manufacturing Systems (NanoCEMMS), University of Illinois, Urbana, Illinois 61801 [8]. MAKING TISSUE ENGINEERING SCAFFOLDS WORK. REVIEW ON THE APPLICATION OF SOLID FREEFORM FABRICATION TECHNOLOGY TO THE PRODUCTION OF TISSUE ENGINEERING SCAFFOLDS E. Sachlos and J.T. Czernuszka* Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, UK [9]. Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture Shawn P. Carey, Casey M. Kraning-Rush, Rebecca M. Williams, Cynthia A. Reinhart-King* Department of Biomedical Engineering, Cornell University, 302 Weill Hall, 526 Campus Road, Ithaca, NY 14853, USA - March 2012 [10]. Hormone-responsive 3D multicellular culture model of human breast tissue Xiuli Wang a, b, David L. Kaplan a, * Biomedical Engineering Department, Tufts University, 4 Colby street, Medford, MA 02155, USA Dalian Institute of Chemical and Physics, Chinese Academy of Sciences, 457, Zhongshan Road, Dalian 116023, China [11]. The use of hyaluronan to regulate protein adsorption and cell infiltration in nanofibrous scaffolds


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Linhao Li a, Yuna Qian a, Chao Jiang a, Yonggang Lv a, Wanqian Liu a, Li Zhong a, Kaiyong Cai a, Song Li b, **,Li Yang a, * Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400030, China Department of Bioengineering, University of California, Berkeley, B108A Stanley Hall, Berkeley, CA 94720-1762, USA - 3 January 2012 [12]. Evolving Three-Dimensional Objects with a Generative Encoding Inspired by Developmental Biology Jeff Clune and Hod Lipson Department of Mechanical and Aerospace Engineering, Cornell University [email protected] [13]. The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1þ enriched skeletal stem cells Manus J.P. Biggs a, *, R. Geoff Richards b, Nikolaj Gadegaard a, Chris D.W. Wilkinson a, Richard O.C. Oreffo c, Matthew J. Dalby a Graphical Models journal homepage: www.elsevier.com/locate/gmod [14]. Procedural function-based modelling of volumetric microstructures q

Alexander Pasko a, Oleg Fryazinov a,⇑ , Turlif Vilbrandt b,c, Pierre-Alain Fayolle d, Valery Adzhiev Bournemouth University, UK Digital Materialization Group, Japan Uformia AS, Norway University of Aizu, Japan Available online 11 March 2011 [15]. Journal of materials science 1 6 (2005) 1121 – 1124 [16]. Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing BARBARA LEUKERS 1 , H ULYA G ULKAN 2 , STEPHAN H. IRSEN 1 , STEFAN MILZ 3 , 1 , MATTHIAS SCHIEKER 2 , HERMANN SEITZ 1 CARSTEN TILLE 1 Research Center Caesar, Ludwig-Erhard-Allee 2, 53175 Bonn 2 Experimental Surgery and Regenerative Medicine, Department of Surgery—Downtown, University of Munich, Nussbaumstrasse 20, 80336 Munchen 3 AO Research Institute, Clavadelerstrasse, 7270 Davos, Switzerland – 2005 [17]. Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds

Yung-Shin Sun,1 Shih-Wei Peng,1,1 Keng-Hui Lin,1,3 and Ji-Yen Cheng1,1,2,a)

Biomicrofluidics. 2012 Marc [18].Development of a new three dimensional cell culture system for the fabrication of artificial epithelial tabule arrays for high content scaffolding – P Flood – 13th of April 2012 – University college Dublin [19].Paper from Bits from Bytes with regards MSDS pla specification sheet – Bits from Bytes - ( A 3d printer Corporation company). [20]. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering S.F. El-Amina, H.H. Lua, Y.Khan a, J.Burems a, J.Mitchell a, R.S. Tuanb,C.T. Laur-encina,c,* Center for Advanced Biomaterials and Tissue Engineering, Department of Chemical Engineering, Drexel University, Room # 383, CAT Building, 3141 Chestnut Street, Philadelphia, PA 19104, USA Depart-ment of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA Department of Orthopae-dic Surgery, Drexel University, College of Medicine, Philadelphia, PA, USA Received 25 May 2002; accepted 28 August 2002 [21]. Natureworks Pla polymer 4032D Biaxially orientated films.pdf – Bits from Bytes – A 3d printer corporation company [22] Biomaterials.2010 May 31(14):3827-39. Epub 2010 Feb 13. - The enhanced characteristics of osteoblast adhesion to photofunctionalized nanoscale TiO2 layers on biomaterials surfaces.Miyauchi T, Yamada M, Yamamoto A, Iwasa F, Suzawa T, Kamijo R, Baba K, Ogawa T.Department of Prosthodontics, School of Dentistry, Showa University, Tokyo, Japan.

mailto:[email protected]

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[23]. Adhesion and Osteoblastic Differentiation of Mouse Mesenchymal Stem Cells Overexpressing Cadherin-11 on Scaffold Surfaces This paper appears in: Bioinformatics and Biomedical Engineering, 2008. ICBBE 2008. The 2nd International Conference on Date of Conference: 16-18 May 2008 Author(s): Shiwu Dong Dept. of Anatomy, Third Mil. Med. Univ., Chongqing Dajun Ying ; Bo Yang ; Zhao Xie ; Xiaojun Duan Page(s): 929 - 932

[24]. MRS conf. proc., Spring 2008, Symposium GG: Mechanical Behavior of Biological Materials and Bioma-terials Proceedings Volume 1097, Epaper 08_GG3-5 Characterisation of Cell Adhesion to Substrate Materials and the Resistance to Enzymatic and Mechanical Cell-Removal Helen J. Griffiths§, John.G. Harvey§, James Dean§, James Curran§, Athina E. Markaki†, T.William Clyne§ §Dept of Materials Science & Metallurgy, Pembroke Street, Cambridge, CB2 3QZ, UK†Dept of Engineering, Trumpington Street, Cambridge, CB2 1PZ, UK [25]. Atomic force microscopy probing of cell elasticity Tatyana G. Kuznetsova a, Maria N. Starodubtseva a,*, Nicolai I. Yegorenkov b, Sergey A. Chizhik c, Renat I. Zhdanov d a Gomel State Medical University, 5, Lange str., Gomel 246000, Belarus b Gomel State Technical University, 48, ave. Oktyabrya, Gomel 246746, Belarus c A.V. Lykov Heat and Mass Transfer Institute of NASB, 15, Brouki str., Minsk 220072, Belarus d Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, 8, Baltiiskaya St., Moscow 125315, Russian Federation 2007 26].http://www.thingiverse.com/thing:15342 : This project used a Next Engine Laser scanner and was undertaken in association with the Pittsburgh Supercomputing Center and the Center for Parabiotics Research. [27].MCF10A and MDA-MB-231 human breast basal epithelial cell co-culture in silicon micro-arrays Mehdi Nikkhah a, b,1, Jeannine S. Strobl b,1, Eva M. Schmelz c, Paul C. Roberts d, Hui Zhou c, Masoud Agah b, * apartment of Mechanical Engineering, Virginia Tech, Blacksburg, VA 24061, USA VT MEMS Lab, The Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg, VA 24061, USA Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA 24061, USA Center for Molecular Medicine and Infectious Diseases (CMMID), Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA 24061, USA - July 2011 [28].Kinetics of bone cell organization and mineralization on materials with patterned surface chemistry Kevin E. Healy*+, Carson H. Thomas * +, Alireza Rezania *t , Jung E. Kim*, Patrick J. McKeownj, Barbara Lom$ and Philip E. Hockberge$ *Division of Biological Materials, Northwestern University Dental School, 37 7 East Chicago Avenue, Chicago, IL of Biomedical Engineering, Robert R. McCormick School of Engineering & Applied Science, Northwestern University, Evanston, IL 60201, USA; t Physical Electronics Incorporated, Eden Prairie, MN55344, USA; 8/institute for Neuroscience, and Departmentof Physiology, Northwestern University Medical School, Chicago, IL 60611, USA - 1996 [29].Layer by Layer Three-dimensional Tissue Epitaxy by Cell-Laden Hydrogel Droplets SangJun Moon, Ph.D.,1,* Syed K. Hasan, M.D.,1,* Young S. Song, Ph.D.,1 Feng Xu, Ph.D., Hasan Onur Keles, B.Sc.,1 Fahim Manzur, B.Sc.,1 Sohan Mikkilineni,1 Jong Wook Hong, Ph.D.,2 Jiro Nagatomi, Ph.D.,3 Edward Haeggstrom, Ph.D.,4 Ali Khademhosseini, Ph.D.,5,6 and Utkan Demirci, Ph.D.1,5,6 - 2009 [30]. Electrospun scaffold topography affects endothelial cell proliferation, metabolic activity, and morphology Daniel E. Heath,1 John J. Lannutti,2 Stuart L. Cooper1

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http://www.thingiverse.com/thing:15342


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Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio 43210 Department of Materials Science and Engineering, The Ohio State University, Columbus, Ohio 43210 Received 26 June 2009; revised 25 November 2009; accepted 4 December 2009 Published online 22 April 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32802 [31].KING TISSUE ENGINEERING SCAFFOLDS WORK. REVIEW ON THE APPLICATION OF SOLID FREEFORM FABRICATION TECHNOLOGY TO THE PRODUCTION OF TISSUE ENGINEERING SCAFFOLDS E. Sachlos and J.T. Czernuszka* Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, UK - 2003 [32].Electrospun Nanofibrous Materials for Neural Tissue Engineering - Yee-Shuan Lee and Treena Livingston Arinzeh * Department of Biomedical Engineering, New Jersey Institute of Technology, Newark, NJ 07102, - USA; E-Mail: [email protected] - * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: 973-596-5269; Fax: 973-596-5222. Received: 31 December 2010; in revised form: 24 January 2011 / Accepted: 28 January 2011 [33]. Biophysical control of invasive tumour cell behaviour by extracellular matrix micro architecture Shawn P. Carey, Casey M. Kraning-Rush, Rebecca M. Williams, Cynthia A. Reinhart-King* Department of Biomedical Engineering, Cornell University, 302 Weill Hall, 526 Campus Road, Ithaca, NY 14853, USA - 2012 [34]. A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration Giorgia Totonelli a, Panagiotis Maghsoudlou a, Massimo Garriboli a, Johannes Riegler Giuseppe Orlando c, Alan J. Burns d, Neil J. Sebire e, Virpi V. Smith e, Jonathan M. Fishman a,Marco Ghionzoli a, Mark Turmaine f, Martin A. Birchall g, Anthony Atala c, Shay Soker c, Mark F. Lythgoe b, Alexander Seifalian h, Agostino Pierro a, Simon Eaton a, Paolo De Coppi a, * Surgery Unit, Institute of Child Health and Great Ormond Street Hospital, University College London, 30 Guilford Street, London WC1N 1EH, UK Centre for Advanced Biomedical Imaging, Department of Medicine and Institute of Child Health, University College London, London WC1N 1EH, UK Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA Neural Development Unit, UCL Institute of Child Health, University College London, London WC1N 1EH, UK Department of Histopathology, Institute of Child Health and Great Ormond Street Hospital, University College London, London WC1N 1EH, UK Division of Bioscience, University College London, London WC1N 1EH, UK UCL Ear Institute, London WC1X 8EE, UK Research Department of General Surgery, Royal Free Hospital, University College London, London NW3 2PF, UK - 2011 [35].The effect of pore size on cell adhesion in collagen-GAG scaffolds F.J. O’Briena,b, B.A. Harleyc, I.V. Yannasc,d, L.J. Gibsona,* Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Rm. 8-135, Cambridge, MA 02139, USA Department of Anatomy, Royal College of Surgeons in Ireland, St. Stephen’s Green, Dublin 2, Ireland Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Rm. 8-135, Cambridge, MA 02139, USA Division of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Rm. 8-135, Cambridge, MA 02139, USA Received 25 November 2003; accepted 16 February 2004 [36].Evolving Three-Dimensional Objects with a Generative Encoding Inspired by Developmental Biology Jeff Clune and Hod Lipson Department of Mechanical and Aerospace Engineering, Cornell University [email protected] - 2011 [37].Poroelasticity of Cartilage at the Nanoscale Hadi Tavakoli Nia,† Lin Han,‡ Yang Li,§ Christine Ortiz,‡ and





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Alan Grodzinsky† Department of Mechanical Engineering, ‡Department of Materials Science and Engineering, §Department of Biological Engineering, Department of Electrical Engineering and Computer Science, and Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts - 2011 [38].Procedural function-based modelling of volumetric microstructures q

Alexander Pasko a, Oleg Fryazinov a,⇑ , Turlif Vilbrandt b,c, Pierre-Alain Fayolle d, Valery Adzhiev Bournemouth University, UK Digital Materialization Group, Japan Uformia AS, Norway - University of Aizu, Japan - 2011 [39]. Characterisation of Cell Adhesion to Substrate Materials and the Resistance to Enzymatic and Mechanical Cell-Removal Helen J. Griffiths§, John.G. Harvey§, James Dean§, James Curran§, Athina E. Markaki†, T.William Clyne§ Dept of Materials Science & Metallurgy, Pembroke Street, Cambridge, CB2 3QZ, UK Dept of Engineering, Trumpington Street, Cambridge, CB2 1PZ, UK - 2008 [40].ypoxia influences the cellular cross-talk of human dermal fibroblasts. A proteomic approach Federica Boraldi a , Giulia Annovi a , Fabio Carraro b , Antonella Naldini b , Roberta Tiozzo

Pascal Sommer c , Daniela Quaglino a,⁎ Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy Department of Physiology, University of Siena, Siena, Italy Institut de Biologie et Chimie des Protéines, CNRS, Université Lyon 1 (UMR 5086), Lyon cedex, France Received 7 March 2007; received in revised form 13 August 2007; accepted 14 August 2007 Available online 22 August 2007 [41].Published online 20 March 2008 | Nature | doi:10.1038/news.2008.675 News - How to print out a blood vessel [42].Hormone-responsive 3D multicellular culture model of human breast tissue Xiuli Wang a, b, David L. Kaplan a, * Biomedical Engineering Department, Tufts University, 4 Colby street, Medford, MA 02155, USA - Dalian Institute of Chemical and Physics, Chinese Academy of Sciences, 457, Zhongshan Road, Dalian 116023, China – 2012[43]. http://courses.washington.edu/conj/bess/bone/bone2.html [44]. The enhanced characteristics of osteoblast adhesion to photofunctionalized nanoscale TiO2 layers on biomaterials surfacesTomohiko Miyauchi a,1, Masahiro Yamada b,1, Akiko Ya-mamoto c,d, Fuminori Iwasa a,b, Tetsuo Suzawa e, Ryutaro Kamijo e, Kazuyoshi Baba a, Takahiro Ogawa b,* a Department of Prosthodontics, School of Dentistry, Showa University, Tokyo, Japan b Laboratory of Bone and Implant Sciences (LBIS), The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, 10833 Le Conte Avenue (B3-087 CHS), Box 951668, UCLA School of Den-tistry, Los Angeles, CA 90095-1668, USA c Biomaterials Center, National Institute for Materials Science (NIMS), Tsukuba, Japan d International Center for Materials Nanoarchitectonics, National Institute for Materials Science (NIMS), Tsukuba, Japan e Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan [45]. TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone Matthew A. Rubin,a Iwona Jasiuk,a,* Jeannette Taylor,b Janet Rubin,c Timothy Ganey,d and Robert P. Apkarianb a The G.W.W. School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0405, USA b Integrated Microscopy and Microanalytical Facility, Department of Chemistry, Emory University, 1521 Pierce Drive, Atlanta, GA 30322, USA c Emory University School of Medicine, Veterans Affairs Medicine Center, 1670 Clairmont Road, Decatur, GA 30033, USA

http://courses.washington.edu/conj/bess/bone/bone2.html


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d Atlanta Medical Center, Department of Medical Education, 303 Parkway Drive NE, Atlanta, GA 30312, USA Received 25 January 2003; revised 12 May 2003; accepted 13 May 2003 [46]. Three dimensional cancellous bone structure in hypoparathyroidism Mishaela R. Rubin a,⁎, David W. Dempster b, Thomas Kohler c, Martin Stauber c, Hua Zhou b, Elizabeth Shane a, Thomas Nickolas a, Emily Stein a, James Sliney Jr. a, Shonni J. Silverberg a, John P. Bilezikian a, Ralph Müller ca Metabolic Bone Diseases Unit, Department of Medicine, College of Physicians and Surgeons, Columbia University New York, NY, USA b Regional Bone Center, Helen Hayes Hospital, West Haverstraw, NY, USA c Institute for Biomechanics, ETH Zurich, Zurich, Switzerland 2010 [47]. Microarchitecture Parameters Describe Bone Structure and Its Strength Better Than BMD Tomasz Topoli ´ nski,1 AdamMazurkiewicz,1 Stanislaw Jung,2 Artur Cicha ´ nski,1 and Krzysztof Nowicki1 [48]. BME504 Tissue Engineering and Regenerative Medicine – Scaffolds Dr George A Burke 1 Faculty of Mechanical Engineering, University of Technology and Life Sciences, Kaliskiego 7 Street, 85-789 Bydgoszcz, Poland 2 South Tyneside Hospital, South Shields, Tyne and Wear NE 34 OPL, UK Correspondence should be addressed to Adam Mazurkiewicz, [email protected] Received 3 November 2011; Accepted 5 December 2011 Academic Editors: E. Tanaka and F. R. Verdun [49]. BSc (Hons) Biomedical Engineering - BME504J2 - Biomaterials & Tissue Engineering Biomolecular Factors in Tissue Engineering - Dr George A Burke Nanotechnology & Integrated Bio-Engineering Centre (NIBEC) School of Electrical & Mechanical Engineering, University of Ulster [50].Topography effects of pure titanium substrates on human osteoblast long-term adhesion 2003 [51]. Single cell adhesion force measurement for cell viability identification using an AFM cantilevere-based micro putter” – Meas Sci Technology – Yajing Shen et al (2011). [52]. Differentiation of Mesenchymal Stem Cells Into Osteoblasts on Honeycomb Collagen Scaffolds Joseph George,1,2 Yoshinori Kuboki,1 Teruo Miyata1 1Koken Bioscience Institute, 2-13-10 Ukima, Kita-ku, Tokyo 115-0051, Japan; telephone: þ81-3-5914-2540; fax: þ81-3-5914-2670; e-mail: [email protected] 2Division of Molecular Medicine, Department of Medicine, Columbia University, 630 West 168th Street, New York, NY 10032 Received 25 January 2006; accepted 13 March 2006 [53].Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing BARBARA LEUKERS1, HU¨ LYA GU¨ LKAN2, STEPHAN H. IRSEN1, STEFAN MILZ3, CARSTEN TILLE1, MATTHIAS SCHIEKER2, HERMANN SEITZ1 1Research Center Caesar, Ludwig-Erhard-Allee 2, 53175 Bonn 2Experimental Surgery and Regenerative Medicine, Department of Surgery—Downtown, University of Munich, Nussbaumstrasse 20, 80336 Mu¨ nchen 3AO Research Institute, Clavadelerstrasse, 7270 Davos, Switzerland 2004 [54]. Adult bone-marrow stem cells and their potential in medicine H T Hassan MD PhD M El-Sheemy MS PhD J R Soc Med 2004;97:465–471 [55]. The enhanced characteristics of osteoblast adhesion to photofunctionalized nanoscale TiO2 layers on biomaterials surfaces.Miyauchi T, Yamada M, Yamamoto A, Iwasa F, Suzawa T, Kamijo R, Baba K, Ogawa TDepartment of Prosthodontics, School of Dentistry, Showa University, Tokyo, Japan. [56]. EM analysis of the nanostructure of normal and osteoporotic human trabecular bone Matthew A. Rubin,a Iwona Jasiuk,a,* Jeannette Taylor,b Janet Rubin,c Timothy Ganey,d and Robert P. Apkarianb [57].Microarchitecture Parameters Describe Bone Structure and Its Strength Better Than BMD - Tomasz Topolinski,1 Adam Mazurkiewicz,1 Stanislaw Jung,2 Artur Cichanski,1 and Krzysztof Nowicki11 Faculty2

http://www.ncbi.nlm.nih.gov/pubmed?term=Miyauchi%20T%5BAuthor%5D&cauthor=true&cauthor_uid=20153521


http://www.ncbi.nlm.nih.gov/pubmed?term=Yamamoto%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20153521

http://www.ncbi.nlm.nih.gov/pubmed?term=Iwasa%20F%5BAuthor%5D&cauthor=true&cauthor_uid=20153521

http://www.ncbi.nlm.nih.gov/pubmed?term=Suzawa%20T%5BAuthor%5D&cauthor=true&cauthor_uid=20153521

http://www.ncbi.nlm.nih.gov/pubmed?term=Kamijo%20R%5BAuthor%5D&cauthor=true&cauthor_uid=20153521

http://www.ncbi.nlm.nih.gov/pubmed?term=Baba%20K%5BAuthor%5D&cauthor=true&cauthor_uid=20153521




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South of Mechanical Engineering, University of Technology and Life Sciences, Kaliskiego 7 Street, 85-789 Bydgoszcz, Poland Tyneside Hospital, South Shields, Tyne and Wear NE 34 OPL, UK Correspondence should be addressed to Adam Mazurkiewicz, [email protected] Received 3 November 2011; Accepted 5 December 2011 [58].Three dimensional cancellous bone structure in hypoparathyroidism Mishaela R. Rubin a,⁎, David W. Dempster b, Thomas Kohler c, Martin Stauber c, Hua Zhou b, Elizabeth Shane a, Thomas Nickolas a, Emily Stein a, James Sliney Jr. a, Shonni J. Silverberg a, John P. Bilezikian a, Ralph Müller Metabolic Bone Diseases Unit, Department of Medicine, College of Physicians and Surgeons, Columbia University New York, NY, USA Regional Bone Center, Helen Hayes Hospital, West Haverstraw, NY, USA Institute for Biomechanics, ETH Zurich, Zurich, Switzerland Revised 24 August 2009 Accepted 19 September 2009 Available online 25 September 2009 Edited by R. Recker [59].MAKING TISSUE ENGINEERING SCAFFOLDS WORK. REVIEW ON THE APPLICATION OF SOLID FREEFORM FABRICATION TECHNOLOGY TO THE PRODUCTION OF TISSUE ENGINEERING SCAFFOLDS E. Sachlos and J.T. Czernuszka* Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, U 2003 [60].Investigations into synthetic and natural inorganic ceramic materials (e.g. hydroxyapatite and tricalcium phosphate)as candidate scaffold material have been aimed mostly at bone tissue engineering ynthetic polymers Aliphatic polyesters such as polyglycolic acid (PGA), polylactic acid (PLLA), their copolymers (e.g. PLGA) and polycaprolactone (PCL) [61]. [60].nanomechanics of the Cartilage Extracellular Matrix Lin Han,1 Alan J.Grodzinsky,2,3,4 and Christine Ortiz Department of Materials Science and Engineering, 2 Department of Electrical Engineering and Computer Science, 3 Department of Mechanical Engineering, and 4 Department of Biological [62].The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes Yasuyo Sugawara a, Ryoko Ando a, Hiroshi Kamioka a, Yoshihito Ishihara a, Sakhr A. Murshid d, Ken Hashimoto b, Noriyuki Kataoka c, Katsuhiko Tsujioka b, Fumihiko Kajiya c, Takashi Yamashiro a, Teruko Takano-Yamamoto d,⁎ a Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medi-cine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata, Okayama-City, Okayama, 700-8525, Japan b Department of Physiology, Kawasaki Medical School, Matushima 57, Kurashiki-city, Okayama, 701-0192, Japan c Department of Medical Engineering, Kawasaki Medical School, Matushima 57, Kurashiki-city, Okayama, 701-0192, Japan d Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai-City, Miyagi, 980-8574, Japan [63].Thermodynamic Model of Short-Term Cell Adhesion In [64].Study of Adhesion Property of Wistar Rat Osteoblasts on Polylactide and MaleicAnhydride Modified-Polylactide PAN Jun WANG Yuan-Liang CAO Xue-Bo SU Lan QIN Jian LU Xiao CAI Shao-Ji 2001

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[66].Potential Use of Stem Cells for Kidney Regeneration

Takashi Yokoo, Kei Matsumoto, and Shinya YokoteProject Laboratory for Kidney Regeneration, Institute of

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DNA Medicine, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461,

Japan Received 11 January 2011; Accepted 18 February 2011 [67] Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture Shawn P. Carey, Casey M. Kraning-Rush, Rebecca M. Williams, Cynthia A. Reinhart-King* Department of Biomedical Engineering, Cornell University, 302 Weill Hall, 526 Campus Road, Ithaca, NY 14853, USA

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Appendix A

Figure 1 - A1 - 24 hours Figure 2 - A1b - 24 hours Figure 3 - combined

Figure 4 - A2a - 24 hours Figure 5 - A2b - 24 hours Figure 6 - combined

41

Investigation of Osteoblast Adhesion on 3d printed scaffolds Jemma Redmond – 09260340 – Msc Nanobioscience

Appendix B

Figure 1 - B92A – 24hr Figure 2 - B92B – 24hr Figure 3 - Combined

Figure 4 - B142A - day 3 Figure 5 - B142B - day 3 Figure 6 - Combined

Figure 7 - b43b - day 3 Figure 8 - b43c - day 3 Figure 9 - Combined

Figure 10 - b44b - day 3 Figure 11 - b44a - day 3 Figure 12 - Combined

Figure 13 - b45a - day 3 Figure 14 - b45b - day 3 Figure 15 - Combined


42

Figure 16 - b45c - day 3 Figure 17 - b45d - day 3 Figure 18 - Combined

Figure 19 - b241a - day 3 Figure 20 - b242b - day 3 Figure 21- Combined



Figure 28 - b112a - day 7 Figure 29- b112b - day 7 Figure 30 - Combined


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Figure 31 - b83d - day 14 Figure 32 - b83c - day 14 Figure 33- Combined

Green shows dyed nucleus of osteoblasts from cell line mc3t3 – sigma aldritch

DAY 1 TOTAL COUNT

AVG DAY 3 TOTAL COUNT



AVGB

B1 10 2 B2 19 3.8 B3 218 43.6 B4 300 60

B5 10 2 B6 47 9.4 B7 137 27.4 B8 190 38

B9 0 0 B10 13 2.6 B11 143 28.6 B12 209 41.8

B13 0 0 B14 50 10 B15 58 11.6 B16 145 29

B17 0 0 B18 12 2.4 B19 110 22 B20 79 15.8

B21 0 0 B22 72 14.4 B23 261 52.2 B24 279 55.8

Table 1 – ‘B’ sample cell count

Figure 34 – B samples over 14 days

Tubles/fibrils Circular Grooves spheres Control Floating

0

10

20

30

40

50

60

70

Sample type

Ce

ll C

ou

nt

(N)

"B" samples over 14 days

24 hours

3 days

7 days

14 days


44

45


Appendix C

Non sanded C1 C7 C13 C19 Sanded left C2 C8 C14 C20 Sanded up C3 C9 C15 C21 Sanded left and up C4 C10 C16 C22 Control C5 C11 C17 C23 Floating Sample C6 C12 C18 C24

Table 1. “C” samples – Sanded and non sanded pla samples


Figure 1 - c91b - day 3 Figure 2 - c91c - day 3 Figure 3 - combined

Figure 4 - c93b - day 3 Figure 5 - c93c - day 3 Figure 6 - - combined

Figure 7 - c101b - day 3 Figure 8 - c101c - day 3 Figure 9 - - combined


46

Figure 10 - c123b - day 3 Figure 11 - c123a - day 3 Figure 12 - combined

Figure 13 - c34a - day 7 Figure 14 - c35b - day 7 Figure 15 - combined

Figure 16 - c36a - day 7 Figure 17 - c37b - day 7 Figure 18 - - combined

Figure 19 -c3a - day 7 Figure 20 - c4b - day 7 Figure 21 - - combined




47

Figure 28 - c12c - day 7





Investigation of Osteoblast Adhesion on 3d printed scaffolds –Jemma Redmond – 09260340 – Msc Nanobioscience

48






49



Figure 57 - c24a - day 14 Figure 58 - x25b - day 14 Figure 59 - combined


50



Figure 64 - c34a - day 14 Figure 65 - c35b - day14 Figure 66 - combined






51


Figure 82 Figure 83 Figure 84- combined

DAY 1 TOTAL COUNT




AVG

C1 47 9.4 C7 11 2.2 C13 256 51.2 C19 248 49.6

C2 52 10.4 C8 25 5 C14 161 32.4 C20 257 51.4

C3 111 22.2 C9 130 26 C15 204 40.8 C21 341 68.2

C4 49 9.8 C10 116 23.2 C16 66 13.2 C22 116 23.2

C5 5 1 C11 18 3.6 C17 345 69 C23 257 51.4

C6 32 6.4 C12 135 27 C18 228 45.6 C24 263 52.6

Table 2 – ‘C’ sample cell count


52

Appendix D

D” samples – Pla – Metacarpal, proximal phalange, middle phalange, distal Phalange bones.

Day 1 D1 D2 D3 D4 D5 D6

Day 3 D7 D8 D9 D10 D11 D12

Day 7 D13 D14 D15 D16 D17 D18

Day 14 D9 D20 D21 D22 D23 D24

Table 1 : Arrangements of “D” samples


Figure 1 - d10a - day 3 Figure 2 - d11b - day 3 Figure 3 - - combined

Figure 4 - d12a - day 3 Figure 5 - d14b - day 3 Figure 6 - combined



53


Figure 13 - d76a - day 3 Figure 14 - d77b - day 3 Figure 15- combined

Figure 16- d81a - day 3 Figure 17 - d82b - day 3 Figure 18 - combined


Figure 22 - d30c - day 7 – uv


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Figure 26- d10a - day 14 Figure 27 - d11b - day 14 Figure 28 - combined





55







56



Figure 59 - d59a - day 14 Figure 60 -d60b - day 14 Figure 61- combined

Figure 61 - d62a - day 14 Figure 62 -d63b - day 14 Figure 63- combined


57

DAY 1 TOTAL COUNT




AVG

D1 24 5.8 D7 142 28.4 D13 118 23.6 D19 59 11.8

D2 22 4.4 D8 107 21.4 D14 100 20 D20 378 75.6

D3 26 5.2 D9 90 18 D15 88 7.6 D21 242 48.4

D4 34 6.8 D10 106 21.2 D16 93 18.6 D22 263 52.6

D5 4 0.8 D11 89 17.8 D17 383 76.6 D23 57 11.4

D6 57 11.4 D12 357 71.4 D18 198 39.6 D24 177 35.4

Table 2 – ‘D’ sample cell count

Table 1 – Appendix D

Metacarpals D1 (24 hours) D6 (3 days) D12 (7 days) D18 (14 days) Proximal phalanges

D2 (24 hours) D7 (3 days) D13 (7 days) D19 (14 days)

Middle phalanges D3 (24 hours) D8 (3 days) D14 (7 days) D20 (14 days) Distal phalanges D4 (24 hours) D9 (3 days) D15 (7 days) D21(14 days) Control D5 (24 hours) D10 (3 days) D16 (7 days) D22 (14 days) Floating Sample D6 (24 hours) D11 (3 days) D17 (7 days) D23 (14 days)

Table 2 – Appendix D-

Metacarpal Proximal phalange

Middle phalange

Distal Phalange

Control Floating

0

10

20

30

40

50

60

70

80

90

Sample types

Ce

ll C

ou

nt

(N)

"D" Metacarpals & Phalanges over 14 days

24 hour

3 days

7 days

14 days


58

Appendix S(E)M

Figure 1 – 200x Figure 2- 200x Figure 3- 200x

Figure 4 - 200x Figure 5 – 200x Figure 6 – 200x

Figure 8 – 200x Figure 9 – 200x Figure 10 – 200x

Figure 11 – 200x Figure 12 – 200x Figure 13 – 200x


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Figure 14- 200x Figure 15 -200x Figure 16- 200x

Figure 17- 200x Figure 18 – 200x Figure 19 - 200x

Figure 30 – 100um pdms Figure 31 – 50um pdms Figure 32- 200um pdms

Figure 33 – 138um pdms Figure 34 – 164um pdms Figure 35 – 150um pdms

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Appendix S

DAY 1 TOTAL COUNT




AVG

S1 Na Na

S2 268 53.8

S3 Na Na

S4 Na Na

Table 1 – ‘S’ sample cell count

Green shows dyed nucleus of osteoblasts from cell line m3tc3 – sigma aldritch

Figure 1 – S4 Figure 2 – S4 Figure 3- combined

Figure 4 – S21a Figure 5 – S21b Figure 6 - combined

Figure 7 – S22a Figure 8 – S22b Figure 9- combined


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Figure 10 – S23a Figure 11 – S23b Figure 12- combined

Figure 13 – s33a Figure 14 – s33b Figure 15 - combined


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An Investigation into Osteoblast Adhesion on 3d printed scaffolds

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