Bone marrow derived mesenchymal stem cell (BM MSC) application in articular cartilage repair 7

Also our results showed that reinforcing the endogenous MSCs by harvesting and local injection to the injured site of the cartilage could lead to a higher quality of cartilage repair as

Moreover, our results showed that the migrated cells in the cartilage defect could increase the quality of the repaired cartilage, which is in accordance to the previous studies by Lee et al (208, 209) Also our results showed that reinforcing the endogenous MSCs by harvesting and local injection to the injured site of the cartilage could lead to a higher quality of cartilage repair as suggested by Fong et al (210)

In conclusion our results indicated that labeling of the cells with an optimized concentration of the SPIO could be a useful tool to evaluate the fate of MSCs after administration It is possible to monitor the migration and localization of

cells using MRI, a non-invasive and repeatable technique, for in vivo

evaluation In addition, we showed that labeled MSCs have the tendency to move to the injured cartilage site, engraft and increase the quality of the repaired cartilage by production of the more hyaline-like cartilage The MSCs also have the tendency to home in the other sources of the inflammation such

as para-patellar fat and surgical scars

Chapter 4 Simulating Injured Articular Cartilage Environment for Mesenchymal Stem Cell Migration Evaluation in A Three Dimensional Microenvironment

4.1 Abstract

Introduction:

Avascular nature of the articular cartilage provides only a limited capacity of self-repair Cell based therapy is one promising approach in the treatment of damaged cartilage Bone marrow (BM) derived mesenchymal stem cell (MSC)

is a good candidate because of their multipotent nature The use of MSCs for cartilage repair relies on the homing and engraftment of the cells to the injured tissue Although there is speculation that injured tissue expresses ligands and chemotactic factors that could attract MSCs, these factors and their

mechanisms are not yet fully understood In vitro modeling is challenging

Microfluidic platforms can study of the cell migration in 3D environment and at the same time provide live observation as well as time laps evaluation of the cellular behavior In this study I designed a microfluidic platform to observe the injured cartilage tissue as well as MSCs at the same time By simulating the injured tissue environment, I could study the effect of MSC on the injured tissue

Purpose:

The purpose of this study is to develop a microfluidic system The system will

be used to evaluate the migration of MSCs against the injured cartilage tissue

Method and approach:

A 3D microfluidic system was developed by integrating a hydrogel scaffold into a polydimethylsiloxane (PDMS) platform, so that it is possible to culture cartilage tissue and MSCs simultaneously The device design was evaluated for the linear concentration gradient of chemo-attractants toward the 3D

hydrogel Also the migration of the MSCs was examined by supplementing the media with platelet-derived growth factor (PDGF) to validate the migration

of the cells Uninjured and injured cartilage tissues were prepared by using an established method Conditioned media were prepared by culturing uninjured and injured cartilage tissues in complete media (CM), and the migration

distance of the cells in conditioned medias and unconditioned CM were

compared The average migration distances of MSCs toward uninjured and injured conditioned media, and tissues were compared RT-PCR were used to investigate expressions of ligand genes such as CXCL10, TGFA, IGF2,

CXCL12, ANGPT1, FGF2, TGFB3, and BMP4, as well as extracellular matrix (ECM) protein genes like COL1A1, and VTN in injured cartilage comparing to the uninjured tissue

Results:

The results showed that MSCs significantly migrated more (in terms of

distance) toward injured cartilage rather than uninjured cartilage The

phenomenon was observed in the movement of cells toward the tissues as well as the conditioned media produced by the tissues

RT-PCR demonstrated that cartilage injury leads to an up-regulation of the gene expressions of collagen type I A1 (COL1A1), chemokine CXC 10

(CXCL10), transforming growth factor-alpha (TGFA), insulin-like growth factor

2 (IGF2), chemokine CXC 12 (CXCL12), angiopoietin 1 (ANGPT1), fibroblast growth factor 2 (FGF2), transforming growth factor beta-3 (TGFβ3), bone morphogenetic protein 4 (BMP4), vitronectin (VTN)

Conclusion:

As I showed in the previous chapter, injection of stem cells in the knee is a promising method for cartilage repair In this chapter I introduced a novel microfluidic platform, which proved to be a flexible tool to study cell migration for various biological applications I confirmed that engraftment of the MSCs in injured cartilage is an active migration and homing process and injured

cartilage encourage the migration of the MSCs toward the injury site I also showed that the cartilage injury up-regulate some specific chemotactic

factors, which can help to find and select a sub-population of MSCs which show stronger response to such factors in cartilage repair Then, on one hand, enhancement of the homing capacity of MSC can be achieved by

modulating their response to chemotactic factors; and on the other hand, modulation can be applied in the site of injury for example with stimulating the target site to attract more MSCs (with releasing more signals)

It provides a well-controlled cell and tissue environment, and real time

monitoring of their interaction Furthermore, it allows for integration of

4.2 Introduction

Cartilage injuries are one of the major causes of disabilities in the world, resulting in substantial morbidity at a high cost for the society (211) The avascular nature of articular cartilage provides a limited capacity of self-

repairing (185, 212, 213) Different approaches include palliative therapy (debridement), microfracture, osteochondral mosaicplasty, and cell based therapy (autologous chondrocyte implantation, or matrix associated cell

implantation)(29) Recently, cell based therapy such as mesenchymal stem cells (MSCs) become one of the promising modalities in treatment of the cartilage injuries (61, 185) and MSCs showed a significant potential for tissue repair (185, 214, 215)

As shown in the previous chapter, the injection of MSCs in the injured

cartilage knee, could improve the quality of the repaired cartilage Presence of the MSCs in the injured cartilage could be due to passive localization or active migration of the cells toward injured cartilage Therefore, to evaluate the effect

of acute cartilage injuries on migration of MSCs, I simulated the MSCs

migration toward the injured cartilage by designing a microfluidic system

Microfluidic platforms are capable of mimicking some of the complexities of in vivo conditions This device provides the opportunity to culture the stem cells

and injured tissues at the same time to observe their interactions The

integrated 3D scaffolds between the microfluidic channels are a simple

imitation of in vivo environment that provide control of the gradient between

channels (178, 216) In addition, high quality imaging capabilities allow for simultaneous real-time monitoring of cells give a better understanding of the

in vivo circumstances (217) Previous studies showed that microfluidic

devices can be used to study the effect of the blocking agents on drug

screening of the epithelial-mesenchymal transition (EMT) phenomenon (218), interactions of the cancer and endothelial cells (219), hepatocyte growth (220) and cell-cell interaction in liver (221, 222), biochemical gradient-guided

cellular dynamics (223, 224) and gradient mediated migration (223), as well

as a simulation on aspects of tissue and organ function (225)

Use of MSCs for cartilage repair relies on the homing and integration of the cells to the injured tissue Although there are speculations that other injured tissues express ligands and chemotactic factors that encourage homing of cells (226-229), but to my knowledge, there is no study which has evaluated the mechanisms of stem cells migration toward injured cartilage and the chemotactic factors secreted by injured cartilage which leads to stem cell migration toward injured cartilage tissue

In this study I developed a microfluidic device to simulate the injured cartilage tissue environment, which provides the ability of simultaneous culturing and monitoring of uninjured cartilage tissue, injured cartilage tissue and MSCs Then, migration of MSCs against the injured cartilage tissue was evaluated Exploring the interaction of MSCs with injured cartilage tissue can open a new avenue in future of cartilage repair strategies

4.3 Methods

On this project, I collaborated with Dr Roger Kamm, head of BioSyM in

SMART institute, Singapore, and his group To be able to culture the cartilage tissue simultaneously with MSCs, I designed and produced a novel

microfluidic device I got trained in BioSyM facilities and performed all the device production and migration experiments to evaluate the stem cells

migration behavior in the presence of the injured and uninjured cartilage tissues To perform a trial on using the microfluidic device as a tool for

studying of stem cell migration, I used one of the established devices at BioSyM The device in figure 4.1 (A) (3-channel device) was designed to evaluate the migration of endothelial cells and angiogenesis (220) and was well described in a manuscript published in peer reviewed journal I got

trained for the microfluidic production and migration assays with help of Dr Amir Aref, a postdoctoral fellow at BioSyM I used the 3-channel device to assess cell migration in the 3D scaffold of microfluidic platform and also to optimize the best collagen polymerization concentration for MSCs As the 3-channel device was not designed for culturing the tissue samples, I needed to design my own device I prepared two different designs of microfluidic

devices The first device was the tissue spider device as shown in figure 4.1B and 4.1C The tissue spider device had some limitations such as risk of cross contamination of the chemotactic factors and the long distance between the tissues, which increased the risk of hypoxia in the center of the collagen channel The second device is the current tissue 3-channel device (figure 4.1D) This device had some advantages to the previous one such as

separate channel for nutrition of each tissue and minimum risk of cross

contamination of the chemotactic factors Also in this design, I used two gel filling channel to minimize the air bubble inside the scaffold Two types of posts secured the tissue area One set of posts which was at the border of collagen filling areas and the media channels was triangular shape and

prevented projection of the scaffold to the media channels The second set of posts, which was inside the collagen filling area to help securing the tissues in place, was columnar to help the distribution of the scaffold in the collagen channel After approval of the design by Dr Kamm, I prepared the AutoCAD map of the device with help of Dr Kim, a postdoctoral fellow at BioSyM The map was sent to Korea for producing the mold After receiving the device mold, I started the production of the microfluidic devices

Figure 4-1 History of microfluidic device design

(A) 3-channel microfluidic device, which is used to perform the pilot migration study (B and C) The tissue spider device template and (D) The tissue 3-channel device were designed for this study (The later one was chosen and produced for the rest of experiments)

4.3.1 Design of microfluidic device

Auto-CAD (Autodesk, CA) was used to design the platform, including the medium channels, tissue chambers, gel filling cages, and micro-pillar

dimensions (figure 4.2) The height of the channels is 250µm and other

dimensions are demonstrated in figure 4.2 In order to culture the tissue, the device was created with one channel at the center for cells and two

semicircular channels at two sides delivering culture medium The collagen gel cages contain the tissue chambers The tissue is embedded in the middle

of each gel cage The side channels are used to deliver the nutrient to the tissue Triangular micro-pillar arrays help the housing of the scaffold in the gel cage and preventing the gel overflow to the channels The round micro-pillar helps to secure the tissue at the same distance from the middle channel in each side By testing different gel concentration the gel cage filling was

optimized The microfluidic channels, tissue chambers and gel cages were cast in polydimethylsiloxane (PDMS), sterilized, and bonded to sterile glass cover after placing the tissue in the device To prevent the microbial

contamination the procedures were done in class II biosafety cabinet The channels were isolated from each other after placing the tissue in the gel chamber and embedding it with the scaffold Tissue can communicate with

of tissue and cells interaction over time Devices were put in 35mm diameter dishes and incubated in 37°C humidified incubator with 5% CO2

Figure 4-2 Schematic design and dimension of microfluidic device

Upper panel shows a schematic image of the tissue microfluidic device Blue channels are the media channels, which are used for control and conditioned media Green channel is the MSCs culture channel Pink channels are the collagen filling area, and tissues will be embedded within the collagen scaffold

in these channels Lower panel shows the Auto-CAD design of the

microfluidic platform; left image demonstrate the dimension of different parts

of the device and right image shows the arrangement of the devices in the master wafer

4.3.2 Computational modeling of concentration gradient

The computational modeling of the device was done with kind help of Dr Kim from BioSyM Gradients of chemotactic factors within the collagen scaffold were quantified by computational modeling using coupled transient

convection-diffusion and Brinkman equations, which were solved with a

commercial finite element solver in COMSOL (Burlington, MA) (230) In

simulations, the diffusion constant of a 40 kDa (average size of factors) inert molecule in the collagen matrix and the diffusion coefficients medium of 6 x 10 -11 m2/s were determined as previously described (230), the factor diffusion coefficient in the scaffold was assumed to be 4.9 x 10 -11 m2/s, taken from the reported values of Helm et al (231) A value of hydraulic permeability (K=10 -13

m2 in the scaffold, where K is the hydraulic permeability of the collagen

matrix) was selected based on reports of Swartz et al (232) Interstitial flow, when applied, was created by imposing a pressure drop of 40 Pa between the central channel and the gel region

4.3.3 Fabrication of microfluidic device

A master silicon wafer was produced by photolithography (233) of the printed Auto-CAD designed microfluidic device transparency mask on SU-8 mold Microfluidic devices were made by repeated molding of PDMS (Dow

Corning® Sylgard 184) on the silicon wafer, curing the polymer by cross-linker

at a ratio of 10:1 (according to the product protocol) and degassed the

elastomer and polymerizing it in 75°C oven for 2 hours Polymerized PDMS was detached from the wafers, each device punched out with a 35mm

diameter puncher, and the inlets were cut out by using 3mm (channel inlets) and 1.2 mm (gel filling inlets) Prior to placing the tissue and bonding the glass