Vascularization in Engineered Tissue Construct by ... - ACS Publications

Subscriber access provided by FONDREN LIBRARY, RICE UNIVERSITY

Article

Vascularization in engineered tissue construct by assembly of cellular patterned micro-modules and degradable microspheres Meiling Zhong, Dan Wei, You Yang, Jing Sun, Xuening Chen, Likun Guo, Qingrong Wei, Yizao Wan, Hongsong Fan, and Xingdong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15697 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

TOC GRAPHIC 112x44mm (300 x 300 DPI)

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Vascularization in engineered tissue construct by assembly of cellular patterned micro-modules and degradable microspheres Meiling Zhong1,2; Dan Wei1; You Yang1; Jing Sun1; Xuening Chen1; Likun Guo1; Qingrong Wei1; * Yizao Wan2, Hongsong Fan1, ; Xingdong Zhang1 1

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, Sichuan, China; 2 College of Materials Science and Engineering, East China Jiaotong University, Nanchang 330013, Jiangxi, China. *Corresponding author: [email protected]; Tel: 86-28-85410703; Fax: 86-28-85410246

KEYWORDS: Cell-laden hydrogel, Micro-modules, Osteon-like, Microfabrication, Vascularization

1


Page 2 of 43

Page 3 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT: Tissue engineering aims to generate functional tissue constructs in which proper ECM for cell survival and establishment of vascular network are necessary. A modular approach via the assembly of modules mimicking the complex tissues’ micro-architectural features and establishing vascular network represents a promising strategy for fabricating larger and more complex tissue constructs. Herein, as a model for this modular tissue engineering, engineered bone-like constructs were developed by self-assembly of osteon-like modules and fast degradable gelatin microspheres. The collagen microspheres acting as osteon-like modules were developed by seeding human umbilical vein endothelial cells (HUVECs) onto collagen microspheres laden with human osteoblast-like cells (MG63) and collagenase. Both HUVECs and MG63 cells were well spatial patterned in the modules, and collagen as ECM well supported cell adhesion, spreading and functional expression due to its native RGD-domains and enzymatic degradation activity. The patterned modules facilitated both the cellular function expression of osteogenic MG63 cells and vasculogenic HUVECs, that is, the osteon-like units were successfully achieved. The assembly of the osteon-like modules and fast degradable gelatin microspheres promoted the vascularization, thus facilitating the osteogenic function expression. The study provides a high-efficient approach to engineer complex 3D tissues with micro-patterned cell types and interconnected channels.

1. INTRODUCTION

A goal of tissue engineering is to generate functional macroscopic tissue construct for 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

replacing lost or damaged tissue.1-3 Conventionally, engineering an artificial tissue involves seeding cells into a preformed three-dimensional (3D) porous scaffold, which has been successful in building a number of tissues for clinical application.4-6 However, challenges still exist in building complex tissues and organs. Recently, a concept of engineering macroscopic tissue constructs based on a so-called “bottom-up” approach has been emerging, and circumvents the limitations of conventional methods.7,8 In this approach, microscopic modules that offer the possibility of mimicking native tissues’ micro-architectural features are assembled into macroscopic tissue constructs.9 Therefore, this modular approach shows a great promise in engineering biomimetic tissue at the mesoscale level. For example, hydrogel fibres encapsulating different cells were assembled to create tissue constructs with cells patterned.10,11 Cell-laden microgels with different shapes and patterned different cells were developed and assembled to imitate osteon or vessel structure.12-14 In these attempts, in addition to the selection of suitable biomaterials for handing process and obtaining construct with desired structure, a most important factor to be considered is the materials functionalization so as to ensure survival and functionality of seeding cells. For the feasibility of incorporating multiple cell types of patterned distribution, hydrogels are most potential to prepare the modules with complex cellular patterns.15 However, to establish the biofunction of the engineered tissues, the hydrogels must be allowed to create proper interface for cell adhesion and suitable space for cell spreading.16 Therefore, besides the prerequisite of proper initial mechanical strength, 3


Page 4 of 43

Page 5 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the design of hydrogel matrix with adhesive sites coupled with sustainable space creating by controlling the degradability (such as enzymatic degradation, chemical degradation, incorporating degradable microspheres) is supposed to be effective to functionalize 3D hydrogels.9,15,17 Apart from the matrix functionalization, the convenience and efficiency to fabricate and assemble the hydrogel modules into meso- or macro-scale structure is another important factor for the “bottom-up” approach. Diverse modular forms have been reported,

including

microfibers,

micropatterned

units,

and

cell-laden

microcarriers.11,18-21 Hydrogel microfibers encapsulating proteins or varied cells can be fabricated by a microfluidic device and woven into macroscopic construct,22 however, the selection of materials for this process is very restricted and the assembly process of using a weaving machine is too complicate to be handled. Micropatterns by photolithography technology show potential for cell patterning and modular design, while the using of UV radiation might impair cells and the assembly process driven by multiphase liquid–liquid tendency or by manual operation is too low-efficiency to be widely used. Nevertheless, cell-laden microcarriers derived from hydrogels show high potential as they can be easily assembled by stacking. For example, Matsunaga et al. prepared collagen gel-based microspheres by microfluidic technology, and after seeding HepG2 cells onto collagen microspheres, they stacked them in a mold and successfully fabricated macroscopic tissue constructs.23 However, the dimension of tissue construct was confined to only several millimeters in size due to the limitation of mass transfer. Herein, another important factor affecting larger tissue survival was 4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

raised, i.e., the construction of vasculature networks in block tissues. It has been acknowledged that an adequate blood supply is necessary for oxygen and nutrient supply in a block tissue as well as a larger engineered tissue construct.24,25 Hence, there is an urgent need of developing high-speed methods to produce biomimetic modules, thus facilitating the convenient assembling process, and creating the vasculature networks within engineered tissues to generate clinically relevant thick tissue substitutes. Osteon is the basic structural unit of bone, and is composed of a Haversian canal containing vasculature surrounded by concentric lamellae of osteocytes. Thus, bone is a good example for the construct of complex tissue. In this work, we used collagen modules containing two types of cells with controllable spatial distribution to generate an osteon-like structure, and explored the biomimetic construction of prevascularized

5


Page 6 of 43

Page 7 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Scheme of construction of macroscopic tissue construct by modular assembly approach: HUVECs-covered MG63-laden collagen modules incorporated gelatin microspheres as modules are assembled randomly into a macroscopic tissue construct.

bone tissue by the assembly of the osteon-like modules. A schematic demonstration of the construction of prevascularized bone tissue is shown in Figure 1. Firstly, human osteoblast-like cells (MG63)-laden hydrogel microspheres were developed, and then human umbilical vein endothelial cells (HUVECs) were seeded on the sphere surface, next, the microspheres were mixed with gelatin microspheres and randomly assembled into a macro-tissue. The microspheres with MG63 cells inner and HUVECs in the sphere outer layer could mimic the complex structure of osteon with both bone part and vascular part. Thanks to the native cellular adhesion sites (RGDs) and the enzyme-cleavable domains of collagen molecules,26-28 the microspheres would offer cell-affinitive domains and create cell spreading space in the presence of collagenase, thus realize the bio-functionalization of the matrix. Due to the fast degradability of gelatin at 37 ℃,9,29 the introduced gelatin microspheres with random distribution in the construct would gradually dissolve over time, thus provide more space among the spherical modules to naturally form vessel-like channels lined with HUVECs. The engineered tissue construct was expected to establish vascular network and enhance osteogenesis.

2. MATERIALS AND METHODS 6



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.1. Materials. Collagen type Ι was extracted from the skin of new-born calf, then purified and dissolved in acetic acid solution. Chondroitin sulfate (CS) was obtained from Haixi Industrial Co. (Shandong, China). Collagenase Type Ι was obtained from Gibco (Cat. no. 17100-017). Gelatin, DAPI (4’,6-diamidino-2-phenylindole), FDA (fluorescein diacetate), PI (propidium iodide), Tetramethyl rhodamine isocyanate (TRITC)-labeled Phalloidin and alizarin red S were purchased from Sigma-Aldrich (USA). Cell TrackerTM CMDil (C7000) and CMFDA (C7025) were obtained from Life technologies Co. (USA). Anti-Osteocalcin antibody (ab13418) and Anti-VE Cadherin antibody (ab33168) were purchased from Abcam (USA). Triton X-100 was obtained from Solarbio Science & Technology Co., Ltd (Beijing, China). Goat serum and secondary antibody were obtained from Zhongshan Jinqiao Biotechnology Co., Ltd (Beijing, China). Pentobarbital sodium was obtained from Sigma (USA). Hematoxylin and Eosin were obtained from Amresco (USA). All other chemicals were acquired from Chengdu Kelong Chem Co. unless otherwise specified. 2.2. Gelatin microspheres fabrication. Gelatin microspheres were prepared with double-emulsion method (w/o/w). Briefly, 2 g gelatin was dissolved in 20 mL PBS at 70 ℃. 10 mL of ethyl acetate was added to 20 mL gelatin solution (10 wt%). After vigorous stirring for 2 min with mechanical stirrer at 700 rpm, the mixture was quickly poured into 60 mL edible oil under agitation at the rate of 300 rpm for 1.5 min. The mixture was then transferred to a cool water bath and maintained at the same stirring rate for 15 min before pouring into ice-cold ethanol for 10 min. Gelatin microspheres were collected by washing with acetone to remove the residual edible 7


Page 8 of 43

Page 9 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


oil, air-dried and separating by standard sieve. Gelatin spheres with diameters of 100-150 µm were utilized for modular assembly. 2.3. Cell culture. MG63 cells and HUVECs were cultured under standard culture conditions. The culture medium was replaced every 2 days and the cells were passaged when confluence was reached.

2.3.1. MG63-laden collagen microsphere fabrication and HUVECs seeding. MG63-laden collagen microspheres were prepared using an electrostatic droplet method as previous reports.17,30 Briefly, collagen in acetic acid solution was firstly adjusted by 5M NaOH to 6.5 at 4 ℃, and then mixed with collagenase. After that, the homogenous blends were centrifuged at 3000 rpm for 2 min to remove entrapped air bubbles. MG63 cells were trypsinized, counted and resuspended in the homogenous blends at a density of 1 x 107 cells per mL, and then the cell containing blends were drawn into a 10 mL syringe. The collagen droplets were prepared by feeding the blends containing cells through nozzles at 5 mL/h, using a syringe pump, while simultaneously exposing them to 7 kV. Then, the collagen droplets were received by the bath containing chondroitin sulfate solution (pH=5.1). As the cell-laden collagen microspheres settled down to the bottom of the bath, the supernatant was replaced with DMEM supplemented with 10% FBS for cell culture. Collagen microspheres with a final collagen concentration of 8 mg/mL and a concentration gradient of collagenase (0, 10, 30 µg/mL) were prepared, named as MS0, MS10 and MS30 respectively. HUVECs at a concentration of 1 x 107 cells per mL of settled collagen 8



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

microspheres were added to MS0, MS10 and MS30 in a 50 mL centrifuge tube and incubated under sterile conditions at 37 ℃ for 6 h with gentle shaking every 30 min, then three modules (MC0, MC10 and MC30) were obtained respectively.

2.3.2. Cell viability and cell morphology observation. At predetermined time points, the samples were stained with FDA/PI and visualized with a confocal laser scanning microscope (CLSM, Leica-TCS-SP5). For actin cytoskeleton staining, the samples were stained with TRITC-labeled phalloidin and counterstained with DAPI according to the manufacturer’s instructions, and the staining process was presented in Supplementary Information, after which the samples were washed in PBS and visualized with CLSM. The morphology of cells after 8 days incubation prepared by critical-point drying was observed using scanning electron microscopy (SEM; JSM-5900LV, Japan). The specimens were coated with gold before observation.

2.3.3. Cell spatial distribution observation. MG63 cells were labeled with CMDil and HUVECs were labeled with CMFDA according to the manufacturer’s instructions, and the staining process was presented in Supplementary Information. Then, the labeled MG63 cells were trypsinized, counted and resuspended in the homogenous blend at a density of 1 x 107 cells per mL during MG63-laden collagen microspheres fabrication process, and the labeled HUVECs were seeded onto the surface of MG63-laden collagen microspheres, and observed under CLSM.

2.3.4. Immunostaining of cells. To examine the osteogenesis of MG63 cells and vasculogenesis of HUVECs respectively, the immunostaining for osteocalcin (OCN) 9


Page 10 of 43

Page 11 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and vascular endothelial cadherin (VECAD) were performed. MC10 were washed three times in PBS, fixed in ice acetone for 15 min, and then rinsed in PBS three times. After that, triton X-100 (0.2%, v/v) was used for permeabilization for 5 min and MC10 were washed in PBS three times, and then blocked with goat serum for 30 min at room temperature. After rinsed in PBS, primary antibody was added for staining overnight incubation at 4 ℃. Then MC10 were rinsed in PBS three times and incubated in second antibody solution for 1h at room temperature under dark condition. Finally, MC10 were rinsed in PBS and incubated in DAPI solution for 5 min to stain the nuclei, after which the samples were washed three times in PBS for confocal imaging. 2.4. Construct assembly. Generally, dry gelatin microspheres were soaked in PBS overnight, then centrifuged. Similarly, MC10 modules in DMEM were let stand for 0.5 h, then the supernatant was sucked out. Finally, MC10 modules and gelatin microspheres at the volume ratio of 5:1 were assembled into a macroscopic bone-like tissue construct in DMEM. As comparisons, MC was prepared by the assembly of MC10 without gelatin microspheres. HUVECs and MG63 cells were simply mixed with pure collagen microspheres (no cells laden, labeled as M10) and gelatin microspheres to obtain MMHG. MMG were prepared by the simple mixture of pure collagen microgels (no cells laden, labeled as M10), gelatin microspheres and MG63 cells. The four constructs are listed in Table 1.

10



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 43

Table 1. Composition of macroscopic tissue constructs and their nomenclature Sample

MC10(mL) (MG63 and HUVECs

M10(mL)

Gelatin

MG63

HUVEs

(No cells)

microspheres

cells/mL

cells/mL

(mL)

patterned)

MCG

0.5

0

0.1

0

0

MC

0.5

0

0

0

0

MMHG

0

0.5

0.1

5x105

5x105

MMG

0

0.5

0.1

5x105

0

2.5. Gene expression. Osteogenesis and vasculogenesis-related gene expression was investigated by a reverse transcriptase polymerase chain reaction (RT-PCR) kit (ToYoBo, Japan). Quantitative real-time PCR reaction was performed by FTC-2000 Real-Time Fluorescence Quantitative Thermocycler (Funglyn Biotech Corp.Ltd, Shanghai, China). The sequences of primers for core-binding factor α1 (Cbfa-1), bone morphogenetic protein-2 (BMP-2), collagen I (COL-I), osteocalcin (OCN), vascular endothelial cadherin (VECAD), platelet endothelial cell adhesion molecule-1 (CD31), human hematopoietic progenitor cell antigen (CD34), von Willebrand factor (vWF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes are given in Supplementary Table S1. Amplification reactions were performed with a SYBR PrimeScript RT-PCR kit (Takara). The mRNA expression level of Cbfa1, BMP-2, Col-I, OCN, VECAD, CD31, CD34, vWF and GAPDH was expressed as threshold cycle (CT) values, and the expression of the housekeeping gene GAPDH was used as 11


Page 13 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


internal control to normalize results. The comparative Ct-value method was used to calculate the relative expression. All samples were analyzed in triplicate. 2.6. In vivo experiments.

2.6.1. Animals and surgical procedure. Twenty-four male BALB/c nude mice (body weight ～ 18 g) were purchased from Chengdu Dashuo Biotechnology Co. Ltd. (Chengdu, China), maintained in a germ-free environment, and allowed free access to food and water. All surgical procedures were conducted according to protocols by the Animal Care and Use Committee of Sichuan University, and the surgery was performed according to the Guide for the Care and Use of Laboratory Animals published by National Academy of Sciences. Macroscopic tissue constructs (MCG, MC, MMHG, MMG) were implanted after in vitro incubation of three days. Six-week-old male BALB/c nude mice were anesthetized by abdominal injection of 0.1% pentobarbital sodium solution (Sigma, USA, 0.1 ml per 25 g body weight) under aseptic conditions. On the back of each nude mouse, two subcutaneous pockets were created and one tissue construct was implanted in each pocket. Four mice were used for per group. Implants were retrieved postoperatively at predetermined time points.

2.6.2. Histological analyses. Animals were sacrificed at various time points, and implants were retrieved. The specimens were fixed in 4% phosphate-buffered paraformaldehyde for 24 h at 4 ℃ and dehydrated in graded ethanol (50%-100%), embedded in paraffin, and then sectioned (5 µm in thickness). To examine the formation of vascular networks and cellular distribution in macroscopic tissue constructs, sections were stained with hematoxylin and eosin (H&E). To examine the 12



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

calcium deposition, specimens were washed twice with PBS, fixed in 95% ethanol and stained with 1% w/v alizarin red S (Sigma). Images were obtained using a phase contrast microscopy (Leica CTR 4000). 2.7. Statistical analysis. All results were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was applied to test the significance of the differences among the study groups. For all statistical tests, the level of significance was set at p < 0.05.

3. RESULTS

3.1. Cell survival and spreading in modules.

3.1.1. MG63 cells in microsphere inner. Figure S1 showed that the electrostatic droplet spray was a high-speed, on-demand production of modules method, and the attained collagen microspheres possessed a large preponderance of live cells (green fluorescence) with high cell density and uniform cell distribution. As shown in Figure 2A, in all the collagen microspheres (MS0, MS10 and MS30), most cells were stained green while little red stained cells presented, and some cells started to form extensions after one-day incubation, indicating live cells had superiority in all collagen the samples. As the culture time prolonged, cells proliferated dramatically, while the number of cells in MS30 was fewer than that in MS10 and MS0. The size of MS30 was smaller than MS10 and MS0 on day 1. Compared with the first day, more cells spread with an increased extension in all collagen microspheres, particularly in MS30 on day 3. Notably, the size of MS10 and MS30 decreased dramatically after 3 days. 13


Page 14 of 43

Page 15 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Moreover, MS started to fuse and formed aggregations due to cell bridging on day 5, and the rate of microsphere fusion increased with the order of MS0 < MS10 < MS30. The cells in MS0 and MS10 showed a typical spindle shape, while cells in MS30 showed a polygonal morphology on day 7. Cellular actin cytoskeleton and cell morphology examined by TRITC-labeled Phalloidin and SEM in Figure 2B and Figure 2C showed that all the cells presented the stretching morphology of immobilized cells, with distinguishable F-actin filaments.

14



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. The morphology of cells in collagen microspheres. (A) Confocal laser scanning microscope (CLSM) images of live (green) and dead (red) MG63 cells encapsulated in collagen microspheres after 1, 3, 5 and 7 days. (B) Actin cytoskeleton of MG63 cells encapsulated in collagen microspheres after 3 days at low magnification and high magnification. (C) SEM images of collagen microspheres at 15


Page 16 of 43

Page 17 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


low magnification and high magnification.

3.1.2. HUVECs on the outer surface of microspheres. The confocal microscopy images of all modules (MC0, MC10 and MC30) with HUVECs seeded are depicted in Figure 3. Almost all cells were stained green (live cells) in all modules and some cells which didn’t adhere on the surface of modules formed cell aggregations after 4 days. The size of modules reduced evidently with the increase of collagenase content. After 7 days, the outer surface of collagen microspheres was covered by a confluent layer of HUVECs with a polygonal morphology, which was consistent with the results of SEM (Figure 3C). Especially, the HUVECs-covered MG63-laden collagen modules encapsulating appropriate collagenase (MC10) showed the most outstanding promotion to the adhesion, proliferation and spreading of HUVECs. Meanwhile, the modules started to fuse after 7 days as shown in Figure 3A. The results of cytoskeleton staining examined with TRITC-labeled Phalloidin in Figure 3B revealed that cells on the surfaces of MC0 and MC10 modules presented a successful reorganization of the cytoskeleton after 7 days, while the filamentous actin (F-actin) cytoskeleton was weaker on the surfaces of MC30 modules.

16



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Cell morphology on the surface of modules. (A) Confocal laser scanning microscope (CLSM) images of live (green) and dead (red) HUVECs spread on the surface of modules after 4 and 7 days. (B) Actin cytoskeleton of HUVECs spread on the surface of modules after 7 days at low magnification and high magnification. (C) SEM images of collagen modules at low magnification and high magnification.

3.2. Cell spatial distribution in osteon-like modules. The successful fabrication of 17


Page 18 of 43

Page 19 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the osteon-like module was further revealed by the visualization of the cell spatial distribution, as showed in Figure 4 with MG63 cells labeled with CMDil red and HUVECs labeled with CMFDA green. The red fluorescence labeled MG63 cells and the green fluorescence labeled HUVECs remained a rounded morphology and located inside and on the surface of MC10, respectively in the initial period. Both MG63 cells and HUVECs proliferated dramatically and spread with an increased extension as culture time prolonged. Notably, MG63 cells were almost completely encased by HUVECs after 7 days.

Figure 4. Fluorescence images showed the cell spatial distribution of MC20. MG63 cells were labeled with CMDil (red) and HUVECs were labeled with CMFDA (green).

3.3. Immunofluorescent staining for specific cellular function expression in osteon-like modules. The expression of osteogenic and angiogenic markers in MC10 18



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was evaluated by immunofluorescent staining, including OCN that was a specific marker of osteogenesis,31,32 and VECAD that was a specific intercellular junction protein of endothelial for vascularization.33,34 As shown in Figure 5, the patterned modules positively expressed both OCN and VECAD proteins after 3 days of cultivation.

Figure 5. Representative immunostaining fluorescence images and light microscopy images of MG63/HUVECs in osteon-like module (MC10). MG63 cells were stained with OCN (green), HUVECs were stained with VECAD (red) and nuclei were stained with DAPI (blue).

3.4. In vitro function expression of the engineered tissue construct. Next, the osteon-like modules were assembled into bone tissue construct and the in vitro function of osteogenesis and vascularization were evaluated by specific gene expression. As shown in Figure 6, there was no significant difference in the levels of Cbfa1 19


Page 20 of 43

Page 21 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Quantitative gene expression of MG63 cells and HUVECs cultured in the engineered macroscopic tissue constructs after 1, 2 and 3 weeks by real-time RT-PCR. (A) Cbfa1, (B) BMP-2, (C) COL-I, (D) OCN, (E) CD31, (F) CD34, (G) vWF and (H) VECAD.

20



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

expression among all the samples at week 1. After 2 weeks, the Cbfa1, BMP-2, COL-I and OCN expression increased in MCG and MC, and a much higher expression was found in MMHG. While in MMG, a low expression was observed. After 3 weeks, gene expression of all the above osteo-markers showed significantly increase in the order of MCG > MMHG > MC. The lowest expression was observed in MMG without HUVECs over the entire culture duration. Notably, the osteogenesis-related gene expression of MCG surpassed that of MMHG at week 3. Simultaneously, expression of vascular markers (CD31, vWF and VECAD) in the engineered tissue constructs - MCG, MC and MMHG showed an obvious increase with the culture time prolonged. For CD34, the highest expression in all samples was found at week 2, and then the expression dropped. In all the time points, gene expression in MC was lower than that in MCG and MMHG, but little difference was found between MCG and MMHG. A low expression was observed over the entire culture duration in MMG without HUVECs presented. 3.5. In vivo evaluation of vascular networks and osteogenesis of engineered tissue construct. To investigate the capacity of the engineered tissue constructs to support vascular networks formation and osteogenesis in vivo, the implanted engineered tissue constructs were characterized by histological staining (Figure 7). H&E staining (Figure 7A) showed the cell spatial distribution and the presence of blood vessels (white arrows) in the engineered tissue constructs. After 2-week implantation, the areas located inside the collagen microspheres and among collagen microspheres were rich with cells in MCG and MC. In contrast, the cells only resided in the spaces 21


Page 22 of 43

Page 23 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. H&E staining and Alizarin red S staining images of removed implants in vivo. Light microscopy images of H&E-stained sections from MCG, MC, MMHG and MMG implants, revealing the presence of numerous blood vessels containing erythrocytes (white arrows) and the osteogenic potential (black arrows) (A). Alizarin red S staining images indicated a large number of calcium nodes (red) at 8 weeks after implantation (B).

among collagen microspheres in MMHG and MMG. It was worth noting that no blood vessel presented in MC and MMG at week 2. However, numerous blood vessels containing erythrocytes were observed to distribute among collagen microspheres of MMHG and MCG, and more blood vessels were found in MMHG. After 4-week implantation, cells proliferated dramatically and located throughout the MCG and MC 22



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

implants. Among all of the implants, there were the largest cell number and best uniform distribution in MCG. In MMHG implants, cells proliferated around collagen microspheres, few cells located in areas of the collagen microspheres. However, few cells and no blood vessels were observed inside the MMG implants, and the cells mainly located on the periphery of implants. After 8-week implantation, cells proliferated dramatically and presented an osteogenic potential in MCG, MC and MMHG. Especially, MCG exhibited the most outstanding osteogenic potential (black arrows), while, in MMG, a small amount of blood vessels appeared and no sign of osteogensis was observed. The mineralization by alizarin red S staining was showed in Figure 7B. After 8-week implantation, there was robust calcium matrix deposition in engineered tissue constructs with the order of MCG>MC>MMHG. Whereas, no positive staining was found in MMG, indicating that no mineralization happened. This osteogenic potential evaluation by mineralization was consistent with the above H&E staining.

4. DISCUSSION

Modular assembly is promising to realize the bio-mimicking of complex tissue from micro-scale to macro-scale based on the principle of bottom-up approach. Two key points must be considered, including cell survival environment and supply of vascular network in block construct. As shown in Figure 1, the module fabrication and assembly in this study possessed the following advantages: (1) the module with both osteoblasts and HUVECs patterned forms an osteon-like unit; (2) the RGD-contained 23


Page 24 of 43

Page 25 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


collagen supports cell adhesion, and the collagenase-induced biodegradation of collagen provides sustained space for cell proliferation and spreading; (3) the natural formed channels among the microspheres due to sphere packing and gelatin degradation form vessel-like channels with HUVECs on the wall, and finally facilitate vascularization inside bone cell-laden matrix. The above results proved the successful realization of the designed functions. The electrostatic spraying technology was confirmed to be efficient to produce cell-laden collagen microspheres and friendly to cells as shown in Figure S1. Based on this, it’s convenient to fabricate cell-laden hydrogel microspheres with high cell density. The following results in Figure 2 and Figure 3 confirmed the successful establishment of cell adhesion and spreading of modules. Almost all of the cells were alive in the microspheres after 1 day indicated the good cytocompatibility of collagen. The better stretched morphology in MS0, MS10 and MS30 in the following days demonstrated a successful reorganization of the cytoskeleton. The good cell survival, spreading and proliferation is no doubt due to the native RGD-domains in the collagen molecules.26,27 While another reason cannot be ignored that the collagen molecules contain enzyme-cleavable domains,27,28 and thus are likely degraded by collagenase, as proven by the size reduction of collagen microspheres. Hence, the faster and better cell spreading exhibited with the increase of collagenase (MS0 MC, notably, the expression in MCG surpassed that in MMHG at week 3. These phenomena suggested that on one hand the interaction of osteoblasts and HUVECs might facilitate the functional expression of osteoblasts, which was corresponding with the previous reports shown by the co-culture of osteoblasts and HUVECs;40,41 on the other hand the patterned modules assembly with both cells might supply the perivascular networks, which could further enhance the osteogenic functional expression. More importantly, the introduction of gelatin microspheres, 26



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

which were easily to be fast degraded at 37 ℃,29 might result in forming space in the modular constructs to facilitate cell proliferation and vascular channels formation. Therefore, the osteogenic expression was the highest in MCG with the patterned arrangement of MG63 cells and HUVECs and the incorporation of gelatin microspheres, while, the lowest expression was found in MMG without HUVECs. As to the vasculogenic function evaluation, CD31, vWF, CD34 and VECAD are all special vascular-markers.42 Similarly, as shown in Figure 6, MCG and MMHG showed better vasculogenic function compared with MC due to the created space among modules via the degradation of gelatin microspheres in engineered tissue constructs. The difference is that the relative higher expression in MMHG than MCG before 2 weeks, which might be thanks to the direct interaction of osteoblasts and HUVECs in MMHG might preferably facilitate the establishment of vasculogenic function in the early stage. Whereas, the patterned HUVECs on modules surface might much facilitate the vascular channels formation, and thus led to the better up-regulating of vasculogenic function of MCG than MMHG in the later stage (at week 3). The above in vitro culture demonstrated the primary function establishment of engineered tissue construct with both osteogenic and pre-vascularized potential. The in vivo analysis further proved that the design of modules, which offered the possibility of mimicking microarchitectural features of native tissues and implementing the prevascularization, played an important role in the functional establishment of engineered tissue constructs (Figure 7). Similarly, MCG and MMHG 27


Page 28 of 43

Page 29 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


significantly promoted the osteogenic and vasculogenic function expression. Furthermore, under the same condition, the direct interaction of osteoblasts and HUVECs was more conducive to the early vascularization compared to the patterned constructs (2w, MMHG>MCG); but the spatially patterned cells in engineered tissue constructs played a more important role in the later vascularization (4w and 8w, MCG>MC>MMHG). Moreover, the created space among modules by degradable gelatin microspheres not only facilitated the osteogenic function expression, but also promoted the vascular network formation (2w and 4w, MCG > MC). Additionally, the vascularization could facilitate the later osteogenesis of engineered tissue constructs. Therefore, the vascularized constructs (MCG, MC and MMHG) exhibited obvious osteogenic potential, and especially MCG with the best vascularization showed the best osteogenic potential. In summary, the osteogenic potential of MC was better than that of MMHG. This might be due to that the osteon-like modules mimicking the microarchitectural features of bone tissue enhanced the vascularization of engineered tissue constructs, thus facilitating the osteogenic potential. However, a small amount of blood vessels appeared and no sign of osteogenic potential was observed in MMG without prevascularization. The results suggest that vascularization plays a significant role in the regeneration of bone tissues, and the design of prevascularized tissue fabrication by modules assembly shows potential for complex tissue construction.

5. CONCLUSION

Successfully

constructing

functional

macroscopic

tissue

28


depends

on

both


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

functionalization of ECM and establishment of vascularization. In this work, functional modules mimicking the osteon-like structures were assembled to realize the biomimetic construction of bone tissue. The cell affinity interface and spreading space, as well as pre-vascularization were successfully established. Collagen microspheres laden with MG63 cells and collagenase in the inner and HUVECs in the outer layer were fabricated to generate osteon-like modules. Then, the bionic bone tissueengineered constructs were obtained by the assembly of osteon-like modules and fast degradable gelatin microspheres, which promoted the vascularization of engineered tissue constructs and further facilitated the osteogenic function expression. The preparation of micropatterned modules combined with the simply bottom-up assembling explored an effective and convenient way to fabricate biomimetic tissue with special functions. This approach is not only potential to engineer complex 3D tissues with 3D micropatterning of different cell types, but also effective to create prevascularized tissue constructs. Future work will focus on applying and adapting the approach towards recapitulating the specific cell arrangements and microenvironment of various tissue types, with the ultimate goal of achieving functional tissue for therapeutic applications.

ASSOCIATED CONTENT

Supporting Information

Actin cytoskeleton staining of cells, preparation of labeled MG63 cells and HUVECs,

29


Page 30 of 43

Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


primers sequences for target genes, preparation of MG63-laden collagen microsphere. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

* Tel.: 86-28-85410703. Fax: 86-28-85410246. Email: [email protected].

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (Contract Grant no. 51473098, 51273121 and 51373105).

REFERENCES

(1) Meyer, U.; Wiesmann, H.P. Tissue engineering: a challenge of today's medicine. Head Face 30



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Med. 2005, 1, 2. (2) Stock, U.A.; Vacanti, J.P. Tissue engineering: current state and prospects. Annu. Rev. Med.

2001, 52, 443-451. (3) Levenberg, S.; Langer, R. Advances in tissue engineering. Curr. Top. Dev. Biol. 2004, 61, 113-134. (4) Langer, R.; Vacanti, J. Tissue engineering. Science. 1993, 260, 920-926. (5) Jithendra, P.; Rajam, A.M.; Kalaivani, T.; Mandal, A.B.; Rose, C. Preparation and characterization of aloe vera blended collagen-chitosan composite scaffold for tissue engineering applications. ACS Appl. Mater. Interfaces. 2013, 5, 7291-7298. (6) Ru, C.; Wang, F.; Pang, M.; Sun, L.; Chen, R.; Sun, Y. Suspended, Shrinkage-Free,

Electrospun PLGA Nanofibrous Scaffold for Skin Tissue Engineering. ACS Appl. Mater.

Interfaces. 2015, 7, 10872-10877. (7) Nichol, J.W.; Khademhosseini, A. Modular Tissue Engineering: Engineering Biological Tissues from the Bottom Up. Soft Matter. 2009, 5, 1312-1319. (8) McGuigan, A.P.; Sefton, M.V. Vascularized organoid engineered by modular assembly enables blood perfusion. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11461-11466. (9) Sun, J.; Wei, D.; Zhu, Y.D.; Zhong, M.L.; Zuo, Y.C.; Fan, H.S.; Zhang, X.D. A spatial patternable macroporous hydrogel with cell-affinity domains to enhance cell spreading and differentiation. Biomaterials. 2014, 35, 4759-4768. (10) Leong, M.F.; Toh, J.K.C.; Du, C.; Narayanan, K.; Lu, H.F.; Lim, T.C.; Wan, A.C.A.; Ying, J.Y. Patterned prevascularised tissue constructs by assembly of polyelectrolyte hydrogel fibres. Nat.

Commun. 2013, 4, 2353-2360. 31


Page 32 of 43

Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(11) Zuo, Y.C.; He, X.; Yang, Y.; Wei, D.; Sun, J.; Zhong, M.L.; Xie, R.; Fan, H.S.; Zhang, X.D. Microfluidic-based generation of functional microfibers for biomimetic complex tissue construction. Acta. Biomater. 2016, 38, 153-162. (12) Du, Y.N.; Ghodousi, M.; Qi, H.; Haas, N.; Xiao, W.X.; Khademhosseini, A. Sequential assembly of cell-laden hydrogel constructs to engineer vascular-like microchannels. Biotechnol.

Bioeng. 2011, 108, 1693-1703 (13) Zuo, Y.C.; Xiao, W.Q.; Chen, X.Q.; Tang, Y.J.; Luo, H.R.; Fan, H.S. Bottom-up approach to build osteon-like structure by cell-laden photocrosslinkable hydrogel. Chem. Commun. 2012, 48, 3170-3172. (14) Zuo, Y.C.; Liu, X.L.; Wei, D.; Sun, J.; Xiao, W.X.; Zhao H.; Guo, L.K.; Wei, Q.R.; Fan, H.S.; Zhang, X.D. Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon. ACS Appl. Mater. Interfaces. 2015, 7, 10386-10394. (15) Wang, C.; Varshney, R.R.; Wang, D.A. Therapeutic cell delivery and fate control in hydrogels and hydrogel hybrids. Adv. Drug. Deliv. Rev. 2010, 62, 699-710. (16) Giancotti, F.G.; Ruoslahti, E. Integrin signaling. Science. 1999, 285, 1028-1033. (17) Zhong, M.L.; Sun, J.; Wei, D.; Zhu, Y.D.; Guo, L.K.; Wei, Q.R.; Fan, H.S.; Zhang, X.D. Establishing a cell-affinitive interface and spreading space in a 3D hydrogel by introduction of microcarriers and an enzyme. J. Mater. Chem. B. 2014, 2, 6601-6610. (18) Wei, D.; Xiao, W.X.; Sun, J.; Zhong, M.L.; Guo, L.K.; Fan, H.S.; Zhang, X.D. A biocompatible hydrogel with improved stiffness and hydrophilicity for modular tissue engineering assembly. J. Mater. Chem. B. 2015, 3, 2753-2763. (19) Chen, M.; Wang, X.; Ye, Z.; Zhang, Y.; Zhou, Y.; Tan, W.S. A modular approach to the 32



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

engineering of a centimeter-sized bone tissue construct with human amniotic mesenchymal stem cells-laden microcarriers. Biomaterials. 2011, 32, 7532-7542. (20) Leferink, A.; Schipper, D.; Arts, E.; Vrij, E.; Rivron, N.; Karperien, M.; Mittmann, K.; Van

Blitterswijk, C.; Moroni, L.; Truckenmuller, R. Engineered micro-objects as scaffolding elements in cellular building blocks for bottom-up tissue engineering approaches. Adv. Mater. 2014, 26, 2592-2599. (21) Chen, M.; Zhou, M.; Ye, Z.; Zhou, Y.; Tan, W.S. Ectopic osteogenesis of macroscopic tissue constructs assembled from human mesenchymal stem cell-laden microcarriers through in vitro perfusion culture. PloS One. 2014, 9, e109214. (22) Onoe, H.; Okitsu, T.; Itou, A.; Negishi, M. K.; Gojo, R.; Kiriya, D.; Sato, K.; Miura, S.; Iwanaga, S.; Shigetomi, K.K.; Matsunaga, Y.T.; Shimoyama, Y.; Takeuchi, S. Metre-long cell-laden microfibres exhibit tissue morphologies and functions. Nat. Mater. 2013, 12, 584-590. (23) Matsunaga, Y.T.; Morimoto, Y.; Takeuchi, S. Molding cell beads for rapid construction of macroscopic 3D tissue architecture. Adv. Mater. 2011, 23, H90-H94. (24) Kannan, R.Y.; Salacinski, H.J.; Sales, K.; Butler, P.; Seifalian, A.M. The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review.

Biomaterials. 2005, 26, 1857-1875. (25) Novosel, E.C.; Kleinhans, C.; Kluger, P.J. Vascularization is the key challenge in tissue engineering. Adv. Drug. Deliv. Rev. 2011, 63, 300-311. (26) Davis, G.E. Affinity of integrins for damaged extracellular matrix: αvβ3 binds to denatured collagen type I through RGD sites. Biochem. Biophys. Res. Commun. 1992, 182, 1025-1031. (27) Ottani , V.; Raspanti, M.; Ruggeri, A. Collagen structure and functional implications. Micron. 33


Page 34 of 43

Page 35 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2001, 32, 251-260. (28) Pilcher, B.K.; Dumin, J.A.; Sudbeck, B.D.; Krane, S.M.; Welgus, H.G.; Parks, W.C. The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix. J.

Cell. Biol. 1997, 137, 1445-1457. (29) Su, K.; Lau, T.T.; Leong, W.; Gong, Y.; Wang, D.A. Creating a Living Hyaline Cartilage Graft Free from Non-Cartilaginous Constituents: An Intermediate Role of a Biomaterial Scaffold.

Adv. Funct. Mater. 2012, 22, 972-978. (30) Shao, W.; Leong, K.W. Microcapsules obtained from complex coacervation of collagen and chondroitin sulfate. J. Biomater. Sci. Polym. Edn. 1996, 7, 389-399. (31) Oh, S.; Brammer, K.S.; Li, Y.S.; Teng, D.; Engler, A.J.; Chien, S.; Jin, S. Stem cell fate dictated solely by altered nanotube dimension. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2130-2135. (32) Huebsch, N.; Arany, P.R.; Mao, A.S.; Shvartsman, D.; Ali, O.A.; Bencherif, S.A.; Feliciano, J.R.; Mooney, D.J. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 2010, 9, 518-526. (33) Dejana, E.; Tournier-Lasserve, E.; Weinstein, B.M. The Control of Vascular Integrity by Endothelial Cell Junctions: Molecular Basis and Pathological Implications. Dev. Cell. 2009, 16, 209-221. (34) Sadr, N.; Zhu, M.; Osaki, T.; Kakegawa, T.; Yang, Y.; Moretti, M.; Fukuda, J.; Khademhosseini, A. SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures. Biomaterials. 2011, 32, 7479-7490. (35) Rho, J.Y.; Kuhn-Spearing, L.; Zioupos, P. Mechanical properties and the hierarchical 34



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 43

structure of bone. Med. Eng. Phys. 1998, 20, 92-102. (36) Ducy, P.; Zhang, R.; Geoffroy, V.; Ridall, A.L.; Karsenty, G. Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation. Cell. 1997, 89, 747-754. (37) Lian, J.B.; Stein, G.S. Concepts of osteoblast growth and differentiation: basis for modulation of bone cell development and tissue formation. Crit. Rev. Oral. Biol. Med. 1992, 3, 269-305. (38) Owen, T.A.; Aronow, M.; Shalhoub, V.; Barone, L.M.; Wilming, L.; Tassinari, M.S.; Kennedy, M.B.; Pockwinse, S.; Lian, J.B.; Stein, G.S. Progressive Development of the Rat Osteoblast Phenotype In Vitro: Reciprocal Relationships in Expression

of

Genes Associated With

Osteoblast Proliferation and Differentiation During Formation of the Bone Extracellular Matrix. J.

Cell. Physiol. 1990, 143, 420-430. (39) Jundt, G.; Berghauser, K.H.; Termine, J.D.; Schulz, A. Osteonectin- a differentiation marker of bone cells. Cell. Tissue. Res. 1987, 248, 409-415. (40) Santos, M.I.; Unger, R.E.; Sousa, R.A.; Reis, R.L.; Kirkpatrick, C.J. Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone–starch scaffold and the in vitro development of vascularization. Biomaterials. 2009, 30, 4407-4415. (41) Unger, R.E.; Ghanaati, S.; Orth, C.; Sartoris, A.; Barbeck, M.; Halstenberg, S.; Motta, A.; Migliaresi, C.; Kirkpatrick, C.J. The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. Biomaterials. 2010, 31, 6959-6967. (42) Bai, H.; Wang, Z.Z. Directing human embryonic stem cells to generate vascular progenitor cells. Gene. Ther. 2008, 15, 89-95.

35


Page 37 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Scheme of construction of macroscopic tissue construct by modular assembly approach: HUVECscovered MG63-laden collagen modules incorporated gelatin microspheres as modules are assembled randomly into a macroscopic tissue construct. 107x72mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. The morphology of cells in collagen microspheres. (A) Confocal laser scanning microscope (CLSM) images of live (green) and dead (red) MG63 cells encapsulated in collagen microspheres after 1, 3, 5 and 7 days. (B) Actin cytoskeleton of MG63 cells encapsulated in collagen microspheres after 3 days at low magnification and high magnification. (C) SEM images of collagen microspheres at low magnification and high magnification. 160x288mm (300 x 300 DPI)


Page 38 of 43

Page 39 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Cell morphology on the surface of modules. (A) Confocal laser scanning microscope (CLSM) images of live (green) and dead (red) HUVECs spread on the surface of modules after 4 and 7 days. (B) Actin cytoskeleton of HUVECs spread on the surface of modules after 7 days at low magnification and high magnification. (C) SEM images of collagen modules at low magnification and high magnification. 160x190mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Fluorescence images showed the cell spatial distribution of MC20. MG63 cells were labeled with CMDil (red) and HUVECs were labeled with CMFDA (green). 160x84mm (300 x 300 DPI)


Page 40 of 43

Page 41 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Representative immunostaining fluorescence images and light microscopy images of MG63/HUVECs in osteon-like module (MC10). MG63 cells were stained with OCN (green), HUVECs were stained with VECAD (red) and nuclei were stained with DAPI (blue). 142x71mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Caption : Figure 6. Quantitative gene expression of MG63 cells and HUVECs cultured in the engineered macroscopic tissue constructs after 1, 2 and 3 weeks by real-time RT-PCR. (A) Cbfa1, (B) BMP-2, (C) COL-I, (D) OCN, (E) CD31, (F) CD34, (G) vWF and (H) VECAD. 160x279mm (300 x 300 DPI)


Page 42 of 43

Page 43 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. H&E staining and Alizarin red S staining images of removed implants in vivo. Light microscopy images of H&E-stained sections from MCG, MC, MMHG and MMG implants, revealing the presence of numerous blood vessels containing erythrocytes (white arrows) and the osteogenic potential (black arrows) (A). Alizarin red S staining images indicated a large number of calcium nodes (red) at 8 weeks after implantation (B). 160x128mm (300 x 300 DPI)


Vascularization in Engineered Tissue Construct by ... - ACS Publications

Recommend Documents