Lin Qi ALL RIGHTS RESERVED

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1 2014 Lin Qi ALL RIGHTS RESERVED

2 POROUS PLGA-CaSiO 3 (PSEUDOWOLLASTONITE) COMPOSITE SCAFFOLDS OPTIMIZED FOR BIOCOMPATIBILITY AND OSTEOINDUCTION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Lin Qi May, 2014

3 POROUS PLGA-CaSiO 3 (PSEUDOWOLLASTONITE) COMPOSITE SCAFFOLDS OPTIMIZED FOR BIOCOMPATIBILITY AND OSTEOINDUCTION Lin Qi Thesis Approved: Accepted: Advisor Dr. Nita Sahai Dean of the College Dr. Stephen Z.D. Cheng Faculty Reader Dr. William J. Landis Dean of the Graduate School Dr. George R. Newkome Department Chair Date Dr. Coleen Pugh ii

4 ABSTRACT The goal of this study was to develop an optimal porous composite scaffold, composed of pseudowollastonite (psw; β-casio 3 ), and poly(l,d-lactic-co-glycolic acid) (PLGA). In culture medium, the psw released Ca 2+ and soluble silica, which are osteoinductive soluble factors. PLGA was utilized as a structural framework and to maintain ph of the scaffold. Scaffolds were prepared by solvent casting/particle leaching technique to obtain porosity and interconnectivity of pores. The mass of NaCl, psw and PLGA were adjusted to determine the effects of the different components on the scaffold s biocompatibility and osteoinductive potential, in vitro, on two types of cells, hmsc (human Mesenchymal Stem Cells) and MC3T3 (murine osteoblast precursor cells). An orthogonal study was designed for 9 scaffold compositions. Scanning electron microscopy and micro-computed tomography showed that the scaffold pore structure depended on the amount of NaCl used during fabrication. Variable porosity, and hydrolysis of the psw and PLGA components resulted in ph changes and different released soluble silica concentrations. Total DNA analysis from cell culture in growth medium showed that scaffold compositions which resulted in a small ph shift (± 0.5 on Day 3) and low soluble silica concentration (65±3 ppm) were less cytotoxic than scaffolds for which ph changes were large (±1.2 on Day 3) and soluble silica concentrations were high (87±5 ppm). The viability of hmscs was iii

5 lower than that of MC3T3 under the same set of conditions. The hmscs were incubated on each scaffold surface for 28 days in osteogenic induction medium and alkaline phosphatase staining was used as a preliminary estimate of osteoinductivity of scaffolds. Two scaffold compositions, which showed lower cytotoxicity and higher osteoinductivity than the other compositions, were selected for LIVE/DEAD staining. Better cell morphology and attachment was observed for the scaffold composed of low NaCl, intermediate psw and intermediate PLGA contents, compared to the scaffold of intermediate NaCl, intermediate psw and high PLGA content. Thus, the optimum psw-plga scaffold composition was determined for cell attachment, viability and a preliminary estimate of osteoinduction. iv

6 ACKNOWLEDGEMENT Foremost, I would like to express my sincere gratitude to my advisor Prof. Nita Sahai and my committee Prof. William J. Landis for their patience, motivation, enthusiasm and immense knowledge. Their educational, professional guidance, comments and advices helped me in all the time of research. Thanks to Prof. Nita Sahai for helping with my thesis when she was extremely busy. I could not have imagined having a better advisor and mentor for my Master study. My sincere thanks also go to Dr. Xianfeng Zhou for all instruction for my experiment and research. His wise and lit ideas were the best inspiration to me. He truly treated and cared me as his little brother. I thank my group members in Prof. Sahai s group: Ziqiu Wang, Weilong Zhao, Zhijun Xu, Steven Mankoci, Hussein Kaddour, Bracamonte A. Guillermo, and Pushkar Sathe for stimulating discussions, for formal seminar rehearsal, for all the fun we have had. I am also grateful to Dr. Bojie Wang for SEM instruction and his motivation. He is the person who truly cared about students. Thanks for logistic support from v

7 Dr. Matthew J. Panzner for use of ICP-OES and Dr. Zhorro Nikolove for use of Micro-CT. I am also thankful to Dr. Xiaohua Yu and Dr. William L. Murphy in University of Wisconsin-Madison. Their advices helped me a lot for my scaffold fabrication. At last but not the least, I would like to thank my family for financial and spiritual support. They care, support, encourage, and instruct me throughout my life. I always believe they are the best parents in the world. I sincere express my best love to them. vi

8 TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION...1 II. MATERIALS AND METHODS Materials Scaffold Fabrication in Cell Culture Well Plate Characterization of Scaffolds by Scanning Electron Microscope Characterization of the Three Dimensional Structure Tomography by X-ray Microtomography Bioactivity Assessment Cell Culture Cell Viability in Growth Medium Total DNA Analysis LIVE/DEAD Staining Cell Differentiation in Osteogenic Induction Medium Statistical Analysis 14 III. RESULTS AND DISCUSSION Bioactive studies Cell viability Osteogenic Differentiation...22 vii

9 3.4 Evaluation of Cell Morphology and Attachment Three-Dimensional Structure of Optimal Scaffold 26 IV. CONCLUSION...28 REFERENCES...30 APPENDIX..32 viii

10 CHAPTER I INTRODUCTION The interdisciplinary field of tissue engineering has already existed for over two decades, requiring researchers to understand areas of biology, material science, chemistry, physics, engineering, geometry, and surgery. [1] The major goal of tissue engineering is to design a scaffold with the functionality, which is proposed to mimicking the reaction of the specific cells to achieve the regeneration of that type of tissue. [2][3] Bone regeneration is required in cases of critical size defects, caused by injury or disease, which do not heal by the body s natural wound-healing process. Three basic components required for bone tissue engineering constitutes are osteogenic cells, a biocompatible scaffold and osteogenic growth factors. [4][5] Polymers such as poly(lactic-co-glycolic acid) (PLGA) has been developed for many years, since the biocompatibility and biodegradability of the materials, and ease in controlling chemical and physical properties. [6][7] PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). PLA is a thermoplastic biocompatible, and biodegradable polymer. [8] However the stiffness (<10 GPa) [9][10] not as high as desired (>10 GPa for human tibia and femur). [11] PGA is also a thermoplastic polymer with high strength and elastic modulus because of its high 1

11 crystallinity, and the degradation rate is faster than that of PLA. [6][12] Thus, PLGA combines the advantages of both PLA and PGA. [6] However when PLGA dissolves by hydrolysis, lactic and glycolic acid are released, result in a decrease in solution ph. [13] This phenomenon will further self accelerate due to acidic ph environment. [6][13] This is an undesired property that limits the application of PLGA. Therefore, a method, which could reduce or neutralize the ph shift, would improve the ability of using PLGA. Silica based bioactive glasses were developed and used clinically in the 1970s. Ceramics, such as calcium silicate, were also found to be osteoconductive. Pseudowollastonite (β-casio 3, psw) is the high temperature polymorph of calcium silicate. Psw dissolves and releases Ca 2+ and soluble silicic acid in simulated body fluid (SBF). In the process, H + are consumed and solution ph increases (Equation 1). CaSiO 3 + 2H + + H 2 O = Ca 2+ + H 4SiO4 (1) The Ca 2+ reacts with PO 3-4 in SBF, a fine surface layer of hydroxyapatite (HAP) is formed. This HAP provides good bond to bone in vivo. Thus, psw is bioactive, or so called osteoconductive (Figure 1). [14]-[17] The osteoinductivity of CaSiO 3 towards hmscs was shown recently. [18] The formation of apatite on psw was faster than on wollastonite (α-casio3, low temperature polymorph) because of a burst release of soluble silica at initial stage of dissolution (peak at Day 1). [19][20] However the fast hydrolytic degradation of psw initially released high concentration of Ca 2+ and silicic acid at levels which were toxic to hmscs. After 3 2

12 days, the dissolution rate decreased, Ca 2+ and silicic acid levels decreased and hmscs proliferated at the psw surface. [18] A limitation of psw and other ceramics is that they are brittle and have poor mechanical strength. 3

13 a) b) Figure 1 a: The cyclic silicate trimer structure within the crystalline structure of psw. The structure plays an important role as active site for stereochemical and initially burst of dissolution. b: Osteogenic gene expressions determined by RT-PCR on psw and wol (wollastonite, α-casio 3 ) at Day 8 and Day 16 in osteogenic induction medium and at a seeding density of 3x10 4 cells ml -1. Genes reported are osteocalcin (OCN), osteopontin (OPN), core-binding factor alpha-1 (Cbfα-1), and β-actin as a house-keeping gene. Four replicates were processed for each condition. hmscs cultured on psw expressed higher osteogenic genes compared with wol, which showed that psw is osteoinductive. Reproduced from [37] with kind permission of the publisher. 4

14 A composite material constituting of PLGA as the framework and psw as the filler would have stiffness and brittleness intermediate between those of PLGA and psw. [6][21]-[23] Further, dissolution of the composite would result in neutralization of the ph drop and ph increase when PLGA and psw are hydrolyzed. [24]-[26] Ideally, the scaffold should mimic the extracellular matrix environment of the osteoblasts, therefore a three-dimensional (porous) scaffold is necessary. [27][28] The pore sizes of nature tissue are ideal for each specific cell to growth and function. [29] The ideal pore size for osteogenic cells is 100 µm to 200 µm. [30]-[32] The scaffolds can be fabricated in various ways, such as solvent casting/particle leaching, using NaCl as the porogen particle to control pore features, such as pore size and interconnectivity. (Figure 2) [33]-[35] The interconnectivity is consisted with the permeability of the scaffold, which contributes to the diffusion of nutrients. [36] The porosity determines the surface area of the scaffold, which in turn will influence its dissolution rate. The released soluble silica ions may inhibit cell viability, but enhance osteogenic diffferentiation. [18][19][37] Therefore, the modulation of cytotoxicity and osteoinductive properties is realized by porous structure. 5

15 6 Figure 2 Schematic of solvent casting/particle leaching method

16 To date, cytotoxicity and osteoinduction studies have been conducted on PLGA [38][39] and psw individually. [19][40] However, the biocompatibility and osteoinductivity for porous PLGA-psw composite have not been studied. The aim of present study is to determine the ideal relative contents of NaCl, psw, and PLGA, which will provide the optimum biocompatibility and osteoinductivity to a three-dimensional, porous composite PLGA-psw scaffold. 7

17 CHAPTER II MATERIALS AND METHODS 2.1 Materials PLGA (molecular weight 20,000 Da) in the ratio of 85:15 was bought from Polyscitech, US. Calcium silicate (particle size ~ 1 µm) and acetone were purchased from Sigma-Aldrich. The porogen, sodium chloride (Sigma-Aldrich), was further separated by diameter size into a range of µm with sieves (VMR). All water used was deionized water (NANOpure diamond TM, Barnstead) with a resistivity of 18.2 MΩ-cm. 2.2 Scaffold Fabrication in Cell Culture Well Plate Three-dimensional porous scaffolds were prepared in a 96-well plate using the solvent casting/particle leaching method. First NaCl and psw crystals were added to the 96-well plate. The plate was placed in a water-jacketed incubator (37 o C, 95% humidity) for 12 hours to ensure salt fusion. The plate was then removed and transferred to a vacuum oven for drying (Thermo Scientific) for 24 hours to eliminate water. PLGA-acetone solution (5 wt.%) was prepared and poured gently into the plate. The plate was covered with PARAFILM and set on ice for 12 hours to make sure that the solution permeated well in the space between NaCl crystals. Holes were poked into the PARAFILM and the plate was left in the hood for 8

18 another 12 hours to evaporate the solvent. Each well was then immersed in deionized water to wash out the salt. The washing water was replaced every 3 hours, the whole washing procedure spent 24 hours. The plate was dried in a vacuum drying oven for 24 hours. After drying, the plate was sealed with PARAFILM and storied in a dry closet. The relative content of NaCl, psw and PLGA was varied in the 96-well plate according to the orthogonal layout shown in Table 1. Three groups were designed in which the relative mass of one component was varied while those of the other two compounds were kept constant. Table 1 Compositions of scaffolds tested in the present study.* The mass of each component is varied and referred to as Low (L), Intermediate (I), or High (H). Mass of NaCl, psw, PLGA (mg) Group 1 Varied NaCl Group 2 Varied psw Group 3 Varied PLGA 80, 2.5, , 2.5, , 2.5, , 0.0, , 2.5, , 5.0, , 2.5, , 2.5, , 2.5, 10.0 *The range of relative mass of each component in scaffolds was selected based on papers. [3][12][27] 9

19 2.3 Characterization of Scaffold Surfaces by Scanning Electron Microscope The scaffolds were removed out of the wells and the surface structure of the scaffolds was characterized by scanning electron microscopy (JEOL, JSM-7401F). The scaffolds were also cut with a razor into small pieces to examine the features of the cut cross-section. 2.4 Characterization of the Three Dimensional Structure Tomography by X-ray Microtomography The three dimensional structure of the scaffold was analyzed by X-ray microtomography (Micro-CT, SkyScan 1172). Spatial resolution was 7 µm for each scanned layer. Scanned layers were reconstructed by software (CTvol and CTvox, SkyScan). The scanned scaffold was the optimal scaffold that selected based on the biocompatibility and osteoinductivities. 2.5 Bioactivity Assessment The scaffolds were immersed in 200 ml simulated body fluid (SBF) each well and maintained at 37 o C. The ion concentrations and ph of SBF are similar to human blood plasma (Table 2). The immersion solutions were collected at day 3, 7, 14, 21, and 28. A ph probe (ORION 9110DJWP, Thermo Scientific) was used to determine ph of each sample. The bioactive silicic acid ions were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). All solution samples were filtered by 0.1µm filter (Cameo) before ICP-OES measurement. 10

20 Table 2 Ion concentrations of the SBF in comparison with that of human blood plasma (mm) Ion Na + K + Mg 2+ Ca 2+ Cl - (HCO 3 ) - (HPO 4 ) 2- (SO 4 ) 2- SBF Plasma Cell Culture For cell growth study, two types of cells, hmsc and MC3T3 (LONZA and ATCC ) were seeded on scaffold surfaces at cell density of 1x10 4 and 3x10 4 cells ml -1, repectively. hmscs were cultured for 3 days in 200 ml growth medium composed of g L -1 Minimum Essential Medium, Alpha 1X (cellgro ), 1% penicillin/streptomycin (Sigma) as antibiotics and 10% fetal bovine serum (Fisher Scientific). MC3T3 were cultured for 3 days in 200 ml growth medium composed of MEM Alpha Modification (1X) (HyClone ), 1% penicillin/streptomycin as antibiotics and 10% fetal bovine serum. For the osteogenic differentiation study, hmscs at passage 4 were cultured at a density of 3x10 4 cells ml -1 for 28 days in the osteogenic induction medium, which was composed of growth medium mm 2-phosphate ascorbic acid nm dexamethasone + 10 mm glycerol 2-phosphate. The medium was changed every 4 days. All cultures were maintained at 37 o C, 5% CO 2 in a water-jacketed humidified incubator. (Thermo Scientific, series II) 11

21 2.7 Cell Viability in Growth Medium Cell growth was determined by total DNA analysis (CyQUANT Cell Proliferation Assay Kit, Invitrogen TM ) and LIVE/DEAD staining (Invitrogen TM ) Total DNA Analysis All the reagents, stock solutions, and DNA standards were provided by the Kit. The experimental protocol for the cell proliferation assay provided with the Kit was used. To prepare the reagent, the concentrated cell-lysis buffer stock solution (Component B) was diluted in distilled water to make 1X cell-lysis buffer. CyQuANT GR working solution (20 ml) was prepared by mixing CyQuANT GR stock solution (Component A) 50 µl and 1X cell-lysis buffer 20mL in 50 ml centrifuge tube thoroughly. For making the DNA standard curve, Bacteriophage λ DNA (200 µl) (Component C) was serially diluted using CyQUANT GR working solution in 2mL centrifuge tube. The concentrations of DNA were modulated by mixing the same volume of standard DNA solution and deionized water. The range of concentrations prepared was from 1000 to 7.8 ng ml -1. To prepare the DNA samples, the scaffolds in the cell culture plate were rinsed twice with phosphate buffered saline (PBS) using pipette. Then cells were then 12

22 lysed using 0.2 ml of 1X cell-lysis buffer. Solutions of lysed cells were transferred into 2 ml centrifuge tubes and centrifuged (eppendorf, Centrifuge 5810 R) at 2000 g for 5 min. The supernatants and standard DNA solutions (50 µl) were then transferred into a 96-well plate. The CyQuANT GR working solution (50 µl) was added to each well, and the samples were incubated at room temperature for 5 min. The fluorescence absorbance of the DNA samples was measured using a fluorescence microplate reader (SYNERGEMX, BioTeK ) with filters at wavelength 480 nm for excitation and 520 nm for emission LIVE/DEAD Staining The cell adhesion, morphology and viability were determined by LIVE/DEAD (Invitrogen TM ) fluorescence imaging. The scaffold surfaces were washed twice with PBS in well plates. Fluorescent agents, calcein AM (calcein acetoxymethyl ester) and EthD-1 (ethidium homodimer) at concentrations of 2 µm and 4 µm respectively were mixed into PBS, and added to the scaffolds for 30 min at room temperature. Cell adhesion, morphology, and viability were then captured by fluorescence microscopy at Day 3. All samples were prepared into triplicate. Calcein AM stained live cells green and EthD-1 stained dead cells red. 13

23 2.8 Cell Differentiation in Osteogenic Induction Medium The enzymatic activity of alkaline phosphatase (ALP) at Day 28 was used as marker of hmsc differentiated down osteogenic lineage. The scaffold surfaces were washed three times with PBS in well plate. 200 ml of 10% buffered formalin (PROTOCOL TM, Fisher Scientific) was then added and incubated at room temperature for 15 minutes to fix the cells. After the cell fixed, scaffolds were washed three times with PBS. Subsequently, nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate (NBT/BCIP 1-step solution, Thermo Scientific) were added and incubated at room temperature for 3 hours for the stain to develop. Finally, scaffolds were washed with PBS in the well plate and dried in room temperature. 2.9 Statistical Analysis Total DNA and ALP staining are conducted on triplicate for each condition. Statistical significances determined by ANOVA test (p<0.05). 14

24 CHAPTER III RESULTS AND DISCUSSION 3.1 Bioactive studies For the variable NaCl group, the concentration of released of soluble silica and ph shift over immersion time in SBF are shown in Figure 3. For initial dissolution of the composite, the high NaCl scaffold reached peak of dissolution with highest soluble silica concentration (~93 ppm at Day 3), compared to the intermediate NaCl (~79 ppm at Day 7) and the low NaCl (~63 ppm at Day 7) scaffolds. Subsequently soluble silica concentration decreased rapidly and reached plateau at ~56, ~70, ~50 ppm, respectively, for the high, intermediate and low NaCl scaffold. The burst released silicic ions caused an initial increase of ph by, and a subsequence rapid decrease of ph as silica concentration decreased. PLGA hydrolysis also contributed to acidic ph shift. The high NaCl scaffold performed like the pure psw scaffold control. (For comparison, bioactive curves for control scaffold: pure psw, pure PLGA, and psw/plga without NaCl are shown in Appendix Figure 1.) The low NaCl scaffold dissolved the slowest with the lowest soluble silica concentration through out the 28 days immersion period. Additionally, the ph of low NaCl scaffold changed least compared to the other two scaffolds. 15

25 Varied NaCl a) b) Varied psw c) d) Varied PLGA e) f) Figure 3 The soluble silica concentration and ph change over immersion time in SBF for all designed scaffolds. The data are organized by the varied component in all the scaffolds. Legend: Low content (L) of NaCl or PLGA and no psw (filled squares) Intermediate content (I) of any component (filled circles) High content (H) of any component (filled triangle) 16

26 The SEM images are shown in Figure 4. From the surface structure of low NaCl and high NaCl scaffolds, the NaCl content dependent phenomenon is explained. The low NaCl scaffold showed a thicker pore wall, while high NaCl scaffold had a thinner pore structure. When the NaCl content increased, the pore wall thickness decreased, and this decreased pore wall thickness raised the contact interface between aqueous phase and scaffold surface, which helped the dissolution for both psw and PLGA. The surface structures of all designed scaffolds were NaCl content dependent, SEM images for the other scaffolds, which had intermediate NaCl content, did not show significant difference for all intermediate NaCl content scaffolds. (Appendix Figure 2) 17

27 a) b) Figure 4 SEM images of scaffolds in the varied NaCl group. (a) low NaCl, intermediate psw, intermediate PLGA (b) high NaCl, intermediate psw, intermediate PLGA 18

28 For variable psw group, the concentration of released of soluble silica and ph shift over immersion time in SBF are shown in Figure 3. The high psw scaffold had the highest soluble silica concentration rapidly (~93 ppm at Day 3, further increased to ~96 ppm at Day 7). This caused the highest ph compared with the other two scaffolds through out immersion period. While the intermediate psw scaffold had a lower soluble silica concentration and ph through out 28 days. The no psw scaffold kept the lowest ph over 28 days, and was finally stable around ph Thus, increased psw in scaffold cased an increase of soluble silica concentration and ph. For variable PLGA group, the soluble silica concentration and ph shift over the period of immersion time in SBF are shown in Figure 3. The low PLGA scaffold showed the highest ph through 28 days, while the high PLGA scaffold shows the lowest ph though 28 days. Lower ph was caused by more PLGA in scaffolds that was available for hydrolysis. After 2 weeks of immersion, the high PLGA scaffold showed a further release of soluble silica (~79 ppm at Day 21 and ~71 ppm at Day 28), which was promoted by lower ph. However, soluble silica concentration of the low PLGA scaffold decreased significantly (~60 ppm at Day 28), although was highest (~81 ppm) at Day 3. It should be noted that the calcium ion did not have significant effect in this study. Calcium concentration over immersion time is shown in Appendix Figure 3. Calcium concentrations were proportionally consisted with soluble silica 19

29 concentration for each scaffold. Generally, calcium concentrations were around the range of 240 ppm, which is the optimum calcium concentration for osteogenic differentiation that has previously verified. [18][41][42] 3.2 Cell Viability Total DNA analysis was done at Day 3 to evaluate the cytotoxicity of each scaffold towards hmscs and MC3T3 cells. All data were collected and sorted based on scaffold groups. (Figure 5) For both cell types in the variable NaCl group, total DNA was decreased with increased NaCl. The cell viability was inversely proportional to soluble silica concentration at Day 3, which is toxic and harmful for cells. [18][19][37] Thus, increase NaCl in scaffolds will raise the interface of scaffold, then cause a higher soluble silica concentration when they dissolved, and further inhibit cell viability. The cell viability was decreased when psw content was increased for both cell types cultured on the variable psw scaffolds. The cell viability was consistent with soluble silica concentrations at Day 3. (0 to 93 ppm) For both the cell lines cultured on the variable PLGA scaffolds, cell viability increased as PLGA content increased. Again, cell viability was consistent with the soluble silica concentrations at Day 3. (82 to 68 ppm) 20

30 a) * * * * b) Figure 5 Total DNA of hmsc (a) and MC3T3 (b) on all scaffolds. Abbreviation L is stand for low, I is for intermediate, H is for high content of each component in the scaffold. *Difference statistically significant (p<0.05) between each scaffold and the I NaCl, I psw, I PLGA scaffold. 21

31 3.3 Osteogenic Differentiation ALP staining was used as a marker for hmscs at Day 28 to evaluate osteoinduction of each scaffold. Cells were incubated in osteogenic induction medium. (Figure 6) For the variable NaCl group, increased NaCl content resulted in decreased osteogenic differentiation. For the variable PLGA group, increasing PLGA content promoted osteogenic differentiation. Differentiation was enhanced by a stable and intermediate soluble silica concentration (56±6 ppm for the variable NaCl group and 74±5 ppm for the variable PLGA group). However, for the variable psw group, both the no psw scaffold and the high psw scaffold showed decreased differentiation compared to the intermediate psw scaffold. These results verify that psw is osteoinductive with initially toxicity. Hence a balance of osteoinduction and its cytotoxicity is important, where the relative content of NaCl/psw/PLGA need to be optimally modulated to obtain optimal and stable soluble silica concentration. [18][37] 22

32 Varied NaCl L (or No)* I H a) b) c) Varied psw d) e) f) Varied PLGA g) h) i) Figure 6 Osteogenic differentiation estimated by ALP staining for all designed scaffolds. hmscs were cultured on each scaffold in osteogenic induction medium for 28 days. Note the greatest ALP activity for the low NaCl content in the varied NaCl group and high PLGA content in the varied PLGA group. * No psw for d) 23

33 3.4 Evaluation of Cell Morphology and Attachment Based on an optimum balance of cell viability and osteogenic differentiation results, two scaffolds were finally selected in each variable content group for evaluating cell morphology and attachment. The low NaCl, intermediate psw, intermediate PLGA scaffold from the variable NaCl group, and intermediate NaCl, intermediate psw, high PLGA scaffold from the variable PLGA group. The scaffold with the best cell viability in the variable psw group was not the best for osteoinduction. Furthermore, the best osteoinductivity one in the psw group (intermediate NaCl, intermediate psw, intermediate PLGA scaffold) was worse than that of the optimal scaffolds in the other two groups, this scaffold was abandoned. To evaluate morphology and attachment of cells, LIVE/DEAD staining was used for both hmscs and MC3T3 cells at Day 3. Two cell lines were incubated in growth medium. (Figure 7) Both cell types attached and expanded better on the low NaCl, intermediate psw, intermediate PLGA scaffold compared to that of the other scaffold, where cells barely expanded. Additionally, the low NaCl, intermediate psw, intermediate PLGA scaffold showed more live cells in green and less dead cells in red compared to the other scaffold. Thus, this scaffold was the optimized scaffold from all the scaffolds in the orthogonally designed study. 24

34 a) b) c) d) Figure 7 LIVE/DEAD staining of hmscs (a,b) and MC3T3 cells (c,d) on two optimal scaffolds at Day 3 in growth medium (cell seeding density was 3x10 4 cells ml -1 for both cell types). The optimal scaffolds were low NaCl, intermediate psw, intermediate PLGA scaffold from the varied NaCl group (a,c) and intermediate NaCl, intermediate psw, high PLGA scaffold from the varied PLGA group (b,d). 25

35 3.5 Three-Dimensional Structure of Optimal Scaffold The optimal scaffold (low NaCl, intermediate psw, intermediate PLGA scaffold) was scanned by X-ray microtomography. (Figure 8) Porosity of scaffold was calculated by the software, the closed porosity was 0.035%, and open porosity was %, which pointed that most of pores were effectively connected. The porosity data verify that the scaffold is high porous and interconnected by solvent casting/particle leaching fabrication method, which promotes the permeability of the scaffold. The porosity for natural cancellous bone is from 30 to 90%. [43] Therefore, the optimal scaffold is suitable for the cancellous bone regeneration. 26

36 2000 µm Figure 8 Micro-CT images for optimal scaffold. The optimal scaffold is the low NaCl, intermediate psw, intermediate PLGA from the varied NaCl group. 27

37 CHAPTER IV CONCLUSION Composite scaffold combined with biocompatible polymers and bioactive ceramics is a promising method for bone tissue regeneration. Porous structure is necessary and important to create an environment mimicking extracellular matrix. We studied different components of scaffolds effect to bioactivity on the level of soluble factors and solution ph, and subsequent osteogenic cells proliferation, osteoinductivity, morphology and adhesion. Increased NaCl content in the scaffolds decreased pore wall thickness, increased the dissolution of psw and PLGA, and showed higher soluble silica concentration. Increased psw content in the scaffold increased soluble silica concentration. Increased PLGA content in the scaffold increased soluble silica concentration, especially after 14 days immersion. The initially higher, cytotoxic Si level caused decreased cell viability in each varied content group. Subsequently, higher osteogenic differentiation showed in scaffolds with intermediate and steady state soluble silica concentration. Cell morphology and adhesion further determined the optimum components content for cell growth. Our results support that the bioactivity is depended on three contents in the scaffolds, and will subsequently effect cell proliferation and 28

38 osteogenic differentiantion. Accounting for the effects of different components contributes towards developing the optimal porous biocompatible, osteoinductive scaffold for bone tissue engineering. 29

39 REFERENCES 1. Langer, R. et al. Tissue engineering, Science Chan, B.P. et al. Eur. Spine J Muneoka, K. et al. Birth Defects Research. Part C Hutmacher, D.W. Biomaterials Fisher, J.P. Functional Tissue Engineering of Bone Zhou, H. et al. Acta Biomater Thomas, V. et al. Curr. Nanosci Raquez, J. M. et al. Biomacromolecules, Leung, L. et al. Biomed. Mater Sodergard, A et al. Prog. Polym. Sci Jae, Y.R., et al. J. Biomechanics Saiful, I.A.R. et al. Intern. J. Basic & Applied Sci Makadia, H.K. et al. Polymers (Basel) De Aza, P. N. et al. J. Microsc De Aza, P. N. et al. J. Dent De Aza, P. N. J. Microsc De Aza, P. N. J Microsc Zhang, N.et al. Biomaterials Zhang, N; PhD thesis, University of Wisconsin-Madison Sahai, N.; Anseau, M. Biomaterials Chandrasekha R. Acta Biomater Anderson, J.M. et al. Adv. Drug Deliv. Rev Zong, X. et al. Biomacromol Bergsma, J.E. et al. Biomaterials Lu, L. et al. J. Biomed. Mater. Res Van der Elst, M. et al. Biomaterials

40 27. Chan, B. P. et al. Eur. Spine J Dorozhkin, S.V. et al. J. Mater. Sci Oh, S.H. et al. Biomaterials Pisanti, P. et al. J. Biomed. Mater. Res. Part A Ishaug-Riley, S. L. et al. Biomaterials Ciara M. et al. Cell Adhesion & Migration Murphy, W. L. et al. Tissue Eng Yang, Y. et al. J. Appl. Polym. Sci Wang, X. et al. J. Mater. Sci. Mater. Med Owen, S.C. et al. Wiley InterScience Zhang, N. et al. Biomater. Sci., Semete, B. et al. Nanomedicine Mura, S. et al. Int. J. Nanomedicine Zhang, N. et al. Birth Defects Res., Part C Maeno, Y. et al. Biomaterials Takagishi, Y. et al. Tissue Engineering John, A.J.W. et al. Acta Biomater

41 APPENDIX The ph and soluble silica concentration evolution against immersion time for controlled scaffolds in SBF are shown in Appendix Figure 1. The ph shift and soluble silica concentration of pure psw curve was consisted with other works. [6][37] The soluble silica concentration initially burst increased and peaked at Day 7, and subsequently began to drop. The ph kept increasing to 7.7. The ph shift of pure PLGA was consisted with related works. [6][17][40] The ph kept dropping over 28 days and finally reached plateau at 6.6. The No NaCl, I psw, I PLGA scaffold showed a delayed release of soluble silica compared to the scaffold with I NaCl. Soluble silica began to significant release after 7 days, increase ph by 0.15 at the same time. 32

42 a) b) Appendix Figure 1 The ph and soluble silica concentration evolution over immersion time for controlled scaffolds in SBF. Controlled scaffolds are No NaCl, I psw, I PLGA (filled squares), pure psw (filled circles), pure PLGA (inversed filled triangles). Curves of the I NaCl, I psw, I PLGA scaffold (filled triangles) are added for comparison. Abbreviation I is stand for intermediate of each content in the scaffold. 33

43 Appendix Figure 2 SEM image of intermediate NaCl content scaffold. It showed a pore wall thickness that intermediate between low NaCl content scaffold and high NaCl content scaffold. 34

44 Appendix Figure 3 Calcium concentration over immersion time for all designed scaffolds. Most of calcium concentrations were ~240 ppm, which is near the optimum range for osteogenic differentiation. [18][41][42] Note: L NaCl scaffold showed the lowest calcium concentration over 28 days, which was proportionally consisted with that of soluble silica concentration. 35