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

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 77   Chapter Simulating Injured Articular Cartilage Environment for Mesenchymal Stem Cell Migration Evaluation in A Three Dimensional Microenvironment 78   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 and to identify potential chemo-attractants for the migration By exploring the interaction of MSCs with cartilage tissue and the chemo-attractant factors, it may be possible to improve current MSC therapies as well as open new avenues for cartilage repair 79   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 80   (IGF2), chemokine CXC 12 (CXCL12), angiopoietin (ANGPT1), fibroblast growth factor (FGF2), transforming growth factor beta-3 (TGFβ3), bone morphogenetic protein (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 biophysical and biochemical factors essential in mimicking physiological conditions for cells after administration Key words: Mesenchymal stem cell (MSC), cell migration, microfluidic device, cartilage, tissue culture 81   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 selfrepairing (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 82   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 83   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 3channel 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 84   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 85     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 3channel device were designed for this study (The later one was chosen and produced for the rest of experiments)   86   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 middle channel through the gel by diffusion and concentration gradients of secreted factors and it receives the nutrition through the lateral semicircular channels The arrangement allows concentration gradient of the chemotactic factors secreted by the tissue toward the cell channel (middle channel) The microfluidic device was designed to fit on the microscope stage for monitoring 87   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 88   simulations, the diffusion constant of a 40 kDa (average size of factors) inert molecule in the collagen matrix and the diffusion coefficients medium of 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 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 cover slips (#1.5 Cell Path, UK), PDMS and glass cover slips were cleaned and autoclaved (20 sterilization and 15 dry) To facilitate the gel filling, devices dried in the 75 °C oven overnight The dried, sterile devices were kept in sterile container and used within days Pre-polymer solution of Collagen type I hydrogel (BD Biosciences Cat No 354236) was prepared in DMEM (Gibco BRL, Grand Island, NY, US), 89   adjusted to pH 7.4 and kept on ice The collagen type I hydrogel were injected through the gel channels till fluid began to touch the triangular pillars Next, the devices were placed in the humidity chambers and incubated in the 37°C humidified incubator for 30 to polymerize the collagen gel To stabilize the collagen gel, complete culture media (DMEM supplemented with 10% FBS (fetal bovine serum) (Gibco BRL, Grand Island, NY, US) and 1% antibiotics (penicillin 100U/ml, streptomycin 0.1mg/ml) (Sigma, St Louis, Missouri, US)) was injected into all three media channels through the channel inlets and incubated overnight All the procedures were done in the class II biosafety cabinet (BSC II) to prevent microbial contamination (contaminated devices were discarded upon detection) 4.3.4 MSC characterization and culture in microfluidic devices Ten milliliter bone marrow was aspirated from iliac crests of mature mini-pig and cultured in the complete media (DMEM supplemented with 10% FBS (fetal bovine serum) (Gibco BRL, Grand Island, NY, US) and 1% antibiotics (penicillin 100U/ml, streptomycin 0.1mg/ml) (Sigma, St Louis, Missouri, US) (each 10mL of bone marrow was cultured in one T-175 flasks) in a humidified atmosphere of 5% CO2, at 37°C After days, non-adherent cells were removed by washing (twice) with PBS and fresh medium was added Cells were cultured to reach 80-90 percent confluency (around 2x106 cells in a T175 culture flask), then, they detached by using TrypLE (Gibco BRL, Grand Island, NY, US) and sub-cultured into two new flasks Cells were expanded and sub-cultured to get enough number of cells for the further experiments (passage 4) The cells were detached and stained with the conjugated antibodies against the hematopoietic, endothelial, and adhesion molecule 90   markers (234)(adhesion molecules CD29, CD44, and CD90; hematopoietic markers CD14, CD34, and CD45; and endothelial marker CD31) Stained cells were characterized by flow cytometry and data acquired using Dakocytomation system and analyzed by Summit SW version 4.3.02 (Beckman Coulter) The differentiation potential of the cells to adipocyte, osteocyte and chondrocyte lineage was confirmed as well (see Chap 4) After characterizing the cells at passage (p3), cells were cultured in the microfluidic devices in a concentration of 100,000 cells/channel to a final confluency of 70-80% 4.3.5 Microfluidic device migration validation Migration were examined by culturing MSCs (passage 3) in complete media and using the supplemented media with platelet derived growth factor (PDGF), a known chemo-attractant for MSCs (235), in the conditioned channel up to days This is used to confirm and validate the capability of our designed microfluidic system to detect cell migration toward the gradient of PDGF as a chemo-attractant factor 4.3.6 Injured and uninjured sample preparation An established ex vivo model was used to prepare the injured and uninjured samples for further experiments (236) Three pairs of circular (6mm diameter) pieces of cartilage tissues (6mm in diameter) were harvested from the female mini-pigs by knee surgery One pair was used for preparation of the cartilage-conditioned media (injured and uninjured) Explants were maintained in culture media for days Then, the media of the samples were changed to fresh media; one piece kept 91   uninjured in the media and the other explant was cut into pieces at 1mm intervals and cultured in the media for 24 hours Then, the media from both explants used for the further experiments (figure 4.3) The second pair was used for preparation of injured and uninjured tissues for microfluidic experiments The explants were cut to small pieces by using a 250µm diameter punch After days maintaining these tissues in media, every two pieces were used in each microfluidic devices One piece as uninjured and the second piece cut into 4-5 smaller pieces as injured samples The third pair of explants was used for the gene expression experiment; one explant was immediately frozen in liquid nitrogen and used as uninjured sample The other one was cut at mm intervals and cultured for 24 hours to be used as injured sample 4.3.7 MSCs migration toward injured cartilage conditioned media Conditioned media were produced by culturing the cartilage tissues in the complete media As described before, two pieces of cartilage from the same porcine knee femoral condyle with the same size (6 mm in diameter) and weight were excised and cultured for days to wash out endogenous chemoattractants (236) After these days, the media of samples exchanged with fresh media and one of these samples was cut to 1x1mm pieces as shown in figure 4.3 and the other sample kept as uninjured sample for further experiments After one day these conditioned media from the both samples were used in six channels (three uninjured conditioned media and three injured conditioned media) to determine the effect of the acute cartilage injuries on the migration of the MSCs (236) The media in the microfluidic 92   devices were replaced daily with conditioned media for days To prevent the tissue dryness, the same amount of the complete media, which used for migration assay were added to the tissues daily   Figure 4-3 Cartilage tissue conditioned media preparation Uninjured (left) and injured (right) cartilage tissue conditioned media preparation 4.3.8 Tissue placement and device assembly As mentioned before pre-polymer solution of Collagen type I hydrogel (BD Biosciences Cat No 354236) was prepared in DMEM (Gibco BRL, Grand Island, NY, US), adjusted to pH 7.4 and kept on ice Pieces of porcine articular cartilage tissues (250 µm thickness) were cut and placed in the tissue chamber and channels were sealed by a sterile cover glass The collagen type I hydrogel were injected through the gel channels till fluid began to touch the triangular pillars To prevent air-bubble and incomplete filling of the cages, two gel filling channels are designed into the device Next, the devices were placed in the humidity chambers and incubated in the 37°C humidified incubator for 30 to polymerize the collagen gel To stabilize the tissue, complete culture media (DMEM supplemented with 10% FBS (fetal bovine serum) (Gibco BRL, Grand Island, NY, US) and 1% antibiotics (penicillin 93   100U/ml, streptomycin 0.1mg/ml) (Sigma, St Louis, Missouri, US)) was injected into all three media channels through the channel inlets and incubated overnight All the procedures were done in the class II biosafety cabinet (BSC II) to prevent microbial contamination (contaminated devices were discarded upon detection) 4.3.9 MSCs migration toward injured tissue Injured cartilage was embedded in the tissue chamber of the microfluidic devices to determine the effect of the tissue on the migration of the MSCs A piece of 1.5 mm in length and 200 µm diameter core of the articular cartilage was placed in one of the tissue chambers of the device and the same size of the cartilage tissue core was cut to 4-5 pieces and placed in the other tissue chamber as the injured tissue and both embedded by the collagen type I scaffold The MSCs were cultured in the middle channel and complete media were added to all channels The migration of the MSCs toward the gradient of chemo-attractants secreted by tissues was evaluated in six devices (six uninjured tissue embedded channels and six injured tissue embedded channels) daily by light microscopy As the migration of the cells toward the tissues was faster than using the conditioned medias, I evaluated the migration till the time that cells reach the tissues, up to days, to exclude the effect of the blockage of the migration by the tissues, as a confounding factor 4.3.10 Quantification of the MSCs migration The average migration distance of MSCs was quantified by measuring the area occupied by MSCs in collagen channel (manually drawn region of interests (ROI)) and dividing the occupied area by the width of collagen 94   channel using ImageJ software version 1.45s, developed by National Institutes of Health (it is showed as “A” in figure 4-7 and 4-9) Each area (A) was calculated for each channel separately (e.g “A Uninjured media“, ”A Injured media“, “A Uninjured tissue“, and “A Injured tissue”) Then, to calculate the average migration distance of the cells in each channel, the calculated areas were divided by length of the base of collagen channel (it is showed as “L” in figure 4-7 and 4-9) Average migration distance = A (Migration area in collagen channel) L (Length of collagen channel base) The calculated distances of MSCs migration against uninjured and injured cartilage tissues were compared and also calculated distances of MSCs migration against uninjured and injured conditioned media were compared For a better visualization of the cells in the collagen channel, the cells migration ROIs were determined by ImageJ software, and an intensity threshold level was established for discrimination between migrated cells and the background The masks of the outlined cells (cells’ masks) were plotted and displayed in the middle panel of figures 4-6, 4-7, and 4-9 4.3.11 Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) Each frozen sample was crushed in the liquid nitrogen using chilled mortar and pestle and suspended in TRIzol reagent (Invitrogen, CA, USA) (236) Crush samples were incubated on ice for 10 minutes with vortex mixing every minutes Chloroform (Invitrogen, Carlsbad, CA, USA) was added to each samples and vortex mixed for minutes The sample is centrifuged at 4oC for 10 minutes at 10,000 rpm The aqueous phase collected and the total RNAs 95   were extracted by using QIAGEN® RNeasy kit The DNase (QIAGEN®, USA) was used to eliminate the possible contamination of DNA The extracted RNA quantity and quality was assessed using NanoDrop spectrophotometer The same amounts of the extracted total RNA of the uninjured and injured articular cartilage samples were used to prepare the complementary DNA (cDNA) with the iScript™ cDNA Synthesis Kit (Biorad, USA) RT-PCR was performed against different chemokines, cytokines, ligands, and growth factors, which could be involved in the MSC migration stimulation according to published literature (Table 3.1) (52, 146, 148-150, 152-154, 157-161, 163, 165, 237-246) Custom-made RT-PCR plate (Applied Biosystems, USA) against the porcine factors was used to compare uninjured and injured samples Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control gene to normalize the results The results were analyzed by 7500 System SDS V1.4.0 software (Applied Biosystems, CA, USA)   96   Table 4-1 List of candidate ligands used for RT-PCR analysis These factors were chosen according to published literatures (52, 146, 148150, 152-154, 157-161, 163, 165, 237-246), which demonstrated their involvement in the MSC migration   Symbol Description   Cxcl 12 Il 1a Il 1b Il Tnf Tgfb1 Tgfb2 Tgfb Bmp2 Bmp4 Bmp7 Fn Vtn Col 1a1 Angpt Angpt2 Vegf a Igf Ihh Igf Fgf Mmp Cx3cl Egf Hbegf Tgf a F2 Ccl Ccl Lif Ntf3 Cxcl 10 Csf Csf Ibsp Chemokine (C-X-C motif) ligand 12 Interleukin 1alpha Interleukin 1beta Interleukin Tumor necrosis factor Transforming growth factor beta Transforming growth factor beta Transforming growth factor beta Bone morphogenetic protein Bone morphogenetic protein Bone morphogenetic protein Fibronectin Vitronectin Procollagen, type 1, alpha Angiopoietin Angiopoietin Vascular endothelial growth factor A Insulin-like growth factor Indian hedgehog homolog, (Drosophila) Insulin-like growth factor Fibroblast growth factor Matrix metallopeptidase Chemokine (C-X3-C motif) ligand Epidermal growth factor Heparin-binding EGF-like growth factor Transforming growth factor alpha Coagulation factor II Chemokine (C-C motif) ligand Chemokine (C-C motif) ligand Leukemia inhibitory factor Neurotrophin Chemokine (C-X-C motif) ligand 10 Colony stimulating factor (gran-macrophage) Colony stimulating factor (granulocyte) Integrin binding bone sialoprotein 97   4.3.11 Statistical analysis Two-way ANOVA with Bonferroni’s multiple comparison adjustment was used to evaluate the statistical differences between the migration distances of the MSCs between groups (e.g., complete media, uninjured and injured cartilage tissue, and respective conditioned medias) A p value of less than 0.05 was considered as significant differences 98   4.4 Results 4.4.1 Computational modeling of concentration gradient Chemo-attractive factors can diffuse into the hydrogel scaffold, mimicking the incorporation of cytokines into the native extracellular matrix (ECM) in the in vivo paradigm Numerical simulations based on a transient solution of the Brinkman equation for porous medium flow and the convection-diffusion equation for factor concentrations demonstrated the development of a nearly linear concentration gradient of growth factors in tissue microfluidic device (Figure 4.4)   Figure 4-4 The growth factors diffusion simulation in 3D scaffold The diffusion simulation toward the 3D scaffold region confirmed the generation of concentration gradient across the collagen matrix, which was nearly linear (C=0 considered as control solution and C=1 as experimental solution which contains growth factor)   99   4.4.2 MSC characterization MSCs were trypsinized from the flasks, stained and analyzed by flow cytometry The cells were positive for adhesion molecules CD29, CD44 and CD90 and negative for hematopoietic markers CD14, CD34 and CD45, and endothelial marker CD31 (Figure 4.5) In addition cells could differentiate to all three adipogenic, osteogenic, and chondrogenic lineage (data showed in chapter 3)   Figure 4-5 Flow cytometry analysis of the stem cells surface markers Harvested cells were positive for CD29, CD44, and CD90, and negative for CD14, CD31, CD45, and CD34   100   4.4.3 Microfluidic device migration validation The devices contained complete media in both channels showed the same migration distance of the MSCs (Figure 4.6A) Longer distances of migration by MSCs were observed in the PDGF conditioned media comparing to control (figure 4.6B) The result confirmed that (a) chemotactic factors can diffuse into the 3D hydrogel scaffold and (b) MSCs will migrate in response to the gradient within the device 101   ... 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). .. Leukemia inhibitory factor Neurotrophin Chemokine (C-X-C motif) ligand 10 Colony stimulating factor (gran-macrophage) Colony stimulating factor (granulocyte) Integrin binding bone sialoprotein 97  ... transforming growth factor beta-3 (TGFβ3), bone morphogenetic protein (BMP4), vitronectin (VTN) Conclusion: As I showed in the previous chapter, injection of stem cells in the knee is a promising