Angiogenesis Potential of Bladder Acellular Matrix Hydrogel by

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The Angiogenesis Potential of Bladder Acellular Matrix Hydrogel by Compounding Endothelial Cells Wenjing Liu, Nailong Cao, Suna Fan, Huihui Zhang, Huili Shao, Lujie Song, Chengbo Cao, Jianwen Huang, and Yaopeng Zhang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00760 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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ACS Applied Bio Materials

The angiogenesis potential of bladder acellular matrix hydrogel by compounding endothelial cells Wenjing Liu1,†, Nailong Cao2, †, Suna Fan1, Huihui Zhang1, Huili Shao1, Lujie Song2,3, Chengbo Cao4,5, Jianwen Huang2,3, *, Yaopeng Zhang1,* 1State

Key Laboratory for Modification of Chemical Fibers and Polymer Materials, International Joint

Laboratory for Advanced Fiber and Low-dimension Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, P.R. China 2Department

of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai

200233, P. R. China 3Shanghai

Eastern Institute of Urologic Reconstruction, Shanghai 200233, China，P. R. China

4School

of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China

5School

of Chemistry and Chemical Engineering, YanTai University, YanTai 264005, PR China

†

These authors contributed equally to this work.

Corresponding Author Yaopeng Zhang, State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, International Joint Laboratory for Advanced Fiber and Low-dimension Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. Tel: +86-21-67792954; Fax: +86-21-67792955; E-mail: [email protected]. Jianwen Huang, MD. PhD. Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, China. Tel: +86-21-64369181; Fax: +86-21-64701361; 1 ACS Paragon Plus Environment

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Email: [email protected].

ABSTRACT Rapid vascularization is very important in tissue-engineering. Bladder acellular matrix (BAM) with inherent bioactive factors, a natural extracellular matrix (ECM) derived biomaterial, has been widely used as a scaffold to facilitate the repair and reconstruction of urinary tissues. However, the application of the traditional BAM scaffold has been limited due to the dense structure. To investigate the angiogenic potential of BAM, BAM hydrogels with tailored porous structures were prepared in this study by tuning BAM concentrations (4, 6 and 8 mg/ml). The 6 mg/ml BAM hydrogel was loaded with porcine iliac endothelial cells (PIECs), and analyzed their angiogenic potential in vitro and in vivo. The mechanical strength and gelation speed of the BAM hydrogels increased, while their pore size decreased with increasing concentration. Commercially available collagen hydrogel (2.5 mg/ml) showed weaker mechanical properties than BAM hydrogels, but similar gelation speed and pore size as 6 mg/ml BAM hydrogel. In order to ensure a similar three-dimensional microenvironment for the PIECs, 6 mg/ml BAM and collagen hydrogels were selected for the in vitro and in vivo experiments. A significantly higher density of viable, fusiform PIECs of average length ~50 μm were observed in the BAM hydrogel, while those inside the collagen hydrogel were spherical and ~30 μm long. In addition, the PIECs/BAM hydrogel resulted in significantly higher revascularization compared with the PIECs/collagen and unloaded BAM hydrogels. The higher angiogenic potential of the PIECs/BAM hydrogel is due to the growth factors that promote PIEC proliferation and therefore vascularization. Keywords: Bladder acellular matrix hydrogel, Collagen hydrogel, Porcine iliac endothelial cells, Growth factors, Angiogenesis

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1 INTRODUCTION Biomaterials derived from the extracellular matrix (ECM) have shown great potential for tissue regeneration in various clinical and preclinical applications1-6 due to their abilities of recruiting progenitor cells,7 and promoting cell migration8-9, proliferation10 and angiogenesis.11 The ECM biomaterials are obtained by removing the cells, while retaining the structural and functional proteins,8,

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glycosaminoglycans (GAGs), and growth factors.13 The bladder acellular matrix (BAM) scaffold has been successfully used for the repair and reconstruction of organs such as the bladder and urethra.14-18 However, traditional two-dimensional BAM scaffold generally exhibits poor adjustability in the preparation process and has nanoscale pores that hinder cell penetration.18-20 In recent years, three-dimensional polymer networks of hydrogels have been extensively used in the tissue-engineering field because of their variable geometry, adjustable strength and porous structure.21 Hydrogel forms of ECM not only can be easily injected into and fill up irregular geometric spaces, but also retain the inherent bioactivity of the native matrix.22-25 In our previous study, we prepared a porous BAM hydrogel by the enzymatic solubilization with pepsin. Endogenous growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and keratinocyte growth factor (KGF) were preserved well in the BAM hydrogel.26 Therefore, the BAM hydrogel has potential uses in tissue engineering as it may obviate the disadvantages of the traditional BAM scaffold. A key issue in tissue-engineering organs is rapid vascularization, which in turn is dependent on the coordinated interplay of different cell types and the above growth factors, that is vital for healing, regeneration and remodeling of the damaged region.27 Several reports have indicated the utility of compounded endothelial cells (ECs) in fibrin gels as models of angiogenesis and vascularization.28-29 Various ECs types have been used to develop models of vascular assembly, especially the mature ECs.28 3 ACS Paragon Plus Environment


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PIEC, as a kind of mature ECs, has been widely co-cultured with other biomaterials in order to demonstrate the effects of vascularization in previous study.26, 30 Furthermore, studies have also reported the significance of VEGF in ECs differentiation and early blood vessel formation, and of PDGF-BB in promoting the maturation of those vessels by recruiting vascular smooth muscles to the nascent capillary-like structures.31-32 Therefore, depending on the coordinated interplay of the ECs and growth factors, BAM hydrogel compounded with ECs may improve the ECs growth in vitro and vascularization in vivo. However, the angiogenesis potential of BAM hydrogel has not been reported. The objectives of this study were: 1) to prepare and characterize the properties of BAM hydrogel including rheology, compression and gelation kinetics, 2) to determine the in vitro pre-vascularization potential of BAM hydrogel by culturing PIECs inside the hydrogel, and 3) to construct PIECs/BAM hydrogel to evaluate the effect of angiogenesis in vivo.

2 MATERIALS AND METHODS 2.1 Experimental design The angiogenic potential of BAM hydrogel with compounding ECs was evaluated in vitro and in vivo. Collagen hydrogel (Rat tail collagen type I, Corning) without growth factors was used as the control. Decellularized BAM sheets from fresh porcine bladder were powdered and dissolved in HCl at different concentrations. The degree of decellularization, and the structure, gelation kinetics and mechanical properties of the resulting BAM hydrogels were determined and compared to that of 2.5 mg/ml collagen hydrogel. To minimize the allogeneic rejection, porcine iliac endothelial cells (PIECs), one of ECs derived from porcine, was used in subsequent in vitro and in vivo experiments. BAM and collagen hydrogels of similar pore sizes were selected for culturing PIECs (PIECs; obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences, China) and their growth and 4 ACS Paragon Plus Environment

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pre-vascularization potential were evaluated. The PIECs compounded BAM (PIECs/BAM) and collagen (PIECs/Col) hydrogels and unloaded BAM hydrogel were then implanted subcutaneously in nude mice to investigate their effects on angiogenesis. 2.2 Decellularization of BAM sheets BAM sheets were obtained from porcine bladder by decellularization as previously described.33-34 Briefly, fresh bladder tissue was rinsed and manually removed muscular and serosal layers, and then treated with 1% (v/v) Triton X-100 (Sigma) and 0.1% (v/v) ammonium hydroxide for 2 weeks to induce cell lysis. To examine the extent of decellularization of the bladder tissue, fresh porcine bladder and decellularized BAM sheets were fixed in 4% paraformaldehyde for 24h, embedded in paraffin, sliced into 5 µm thick serial sections and stained with hematoxylin and eosin (H&E) to detect intact nuclei. Masson trichrome was then used to assess the change in collagen architecture before and after decellularization. All stained sections were observed by optical microscopy (DS-Ri1, Nikon, Tokyo, Japan) 2.3 Digestion and solubilization of BAM Decellularized porcine bladder sheets were lyophilized and ground into a fine powder with a cryogenic mill (FREEZER/MILL 6770, SPEX Sample Prep), and stored at -50 °C until further use. The digestion protocol of BAM was modified from previously described methods.35-36 Briefly, 100 mg BAM powder and 10 mg pepsin (Sigma) were separately deposited into 10 ml of 0.01 M HCl solution and stirred at room temperature for 48 h at 150 rpm. The resulting BAM digests (~pH 2) at the concentration of 10 mg/ml were stored at 4 °C until use. 2.4 Preparation of BAM and collagen type I hydrogels BAM hydrogels were prepared from the digests as previously described.25-26, 37 Briefly, the BAM digests were neutralized by adding 1/10th the volume of cold (4 °C) 0.1 N NaOH and 1/9th of that of cold 10×



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PBS to prepare an isotonic pre-gel solution. Different concentrations (4, 6, 8 mg/ml) of this solution were diluted with cold 1× PBS and then incubated for 30 min at 37°C to induce hydrogel formation. Collagen type I hydrogel at the concentration of 2.5 mg/ml was prepared as per the manufacturer’s instructions. All pre-gel solutions were stored at 4°C and used within 24h. 2.5 Scanning electron microscopy (SEM) The internal pore structure of the BAM hydrogels was examined by SEM (Quanta 250, Czech Republic) in order to determine the concentration which had a similar internal pore structure as the 2.5 mg/ml collagen hydrogel. Briefly, 500 l pre-gel solution was poured in a 24-well plate and polymerized at 37°C for 1h. The resulting hydrogel samples were frozen and lyophilized at -50°C for 24h. Subsequently, the dried hydrogel samples were quenched in liquid nitrogen to obtain cross sections for SEM, and images were taken to compare the pore structures of different hydrogels. 2.6 Hydrogel gelation kinetics analysis The gelation kinetics of BAM and collagen hydrogels were analyzed and compared by observing the changes in turbidity, which was measured as previously described.36,

38

Neutralized 4, 6 and 8 mg/ml

BAM and 2.5 mg/ml collagen pre-gel solutions were prepared on ice, and 100 l of each solution was added per well in triplicates in 96-well plates. Optical density (OD) was read every 2 min at 405 nm for 50 min using an ultra-micro spectrophotometer (Multiskan GO, Thermo Scientific) at 37°C. The OD curves were obtained, and normalized absorbance (NA) curves were further calculated according to the following equation: 𝑁𝐴 =

𝐴 ― 𝐴0 𝐴𝑚𝑎𝑥 ― 𝐴0

where A is the reading value at a measured time, A0 is the initial reading value and Amax is the maximum reading value. The gelation kinetic parameters such as gelation speed (S), t lag, and t 1/2 were extrapolated 6 ACS Paragon Plus Environment

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according to the NA curves. S was calculated as the maximum slope of the growth portion of the curve, t lag as

the intercept of the linear region of gelation, and t 1/2 was defined as the time to reach 50% gelation.36,

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2.7 Analysis of rheological behavior and compressive properties The rheological properties of BAM and collagen hydrogels were determined by a Rotational Rheometer (RS150L, HAAKE Instruments), using a 35 mm diameter plate with a 0.3 mm gap at 37 °C. Initially, a 30 min oscillatory time sweep was performed at 1% strain and 1 rad/s (0.1592 Hz) to induce gelation of all samples. Gelation was considered complete when the G’ and the G’’ increased to steady equilibrium values without significant change. The maximum G’ of all samples were then averaged (with standard deviation) to compare the elasticity of the hydrogels as previous studies.25, 39 Five samples per gel were tested. Immediately afterwards, an oscillatory frequency sweep (frequency from 0.1 to 100 rad/s, 1% strain) and an oscillatory amplitude sweep (strain covering the range 0.1–100%, 1 rad/s frequency) were performed, respectively. Compressive tests were used to further characterize the mechanical properties of the hydrogels. Briefly, 900 l pre-gel solutions were poured into 12 mm diameter cylinder molds and incubated at 37 °C for 60 min to form solid hydrogels. The hydrogel cylinders (height 6-8 mm, 12 mm diameter) were tested in triplicates on a universal testing machine (MTS Exceed E42) with a 25 N load cell at a cross-head speed of 1 mm/min until failure. Broken strength and compressive moduli were obtained according to the compression curves. Broken strength is defined as the compressive strength at hydrogel failure, while compressive modulus is defined as the maximum slope of compression curves. 2.8 Construction of PIECs/hydrogels In order to assess the pre-vascularization potential of BAM hydrogel in vitro, PIECs were cultured inside



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6mg/ml BAM and 2.5mg/ml collagen hydrogels. Briefly, PIECs suspensions were mixed with BAM or collagen pre-gel solutions at the final cell density of 1.5×105 cell/ml to obtain cell-loaded pre-gel solutions. Stainless steel annular rings (inner diameter: 1.15 cm, outer diameter: 1.5 cm) were placed inside the wells of a 24-well culture plate, and 300 l of these solutions were injected per well over the rings. The plates were incubated at 37 °C for 45 min to form a cell-loaded hydrogel. The wells were then filled with DMEM containing 10% fetal bovine serum (FBS) and 1% streptomycin, and the steel rings were removed. The culture medium was changed every 2 days. 2.9 Evaluation and comparison of PIEC growth inside hydrogels The viability of the cells inside the BAM and collagen hydrogels was determined using a Live/Dead Viability Cytotoxicity Assay Kit (KeyGEN BioTECH) on day 7 post-loading. Briefly, 500 µl of the kit staining solution containing 2 mM Calcein AM and 8 mM Propidium Iodide (PI) was directly added onto the gel and incubated for 45 min, which were then observed by Laser Scanning Confocal Microscope (LSCM, TCS SP5Ⅱ). Compressed 3D z-stacks images were taken and 3D-cube images were simulated by Imaris X6 software. The effect of BAM hydrogel PIEC growth in vitro was also analyzed in terms of the cytoskeletal structure of PIECs after 7 days of culture by staining the cells with phalloidin (Cytoskelton, USA) which dyed the cytoplasm red, and DAPI (4’,6-diamidino-2-phenylindole, Sigma) which dyed the nucleus blue. Briefly, PIECs/hydrogels were immersed in 4% paraformaldehyde for 10 min and treated with 0.1% (v/v) Triton-X 100 and 1% (v/v) BSA for 5 min and 30 min respectively at room temperature. Subsequently, phalloidin and DAPI were used to stain the cells for 30 min and 10 min respectively. The length of PIECs were quantitatively measured using their fluorescent images by Nano Measurer 1.2. H&E staining as a histological method was used to directly observe the proliferation and distribution of


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PIECs inside hydrogels on day 7 of culture.25 Briefly, the PIECs/hydrogels were fixed in 4% paraformaldehyde for 24 h, rinsed with water and dehydrated in an alcohol gradient, and finally embedded in paraffin. Cross sections and maximum sections were separately sliced and stained, and then observed by optical microscopy (DS-Ri1, Nikon, Tokyo, Japan). 2.10 Scaffold implantation in vivo All animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital prior to study initiation. To avoid immunological rejection which resulted from heterologous PIECs in vivo, nude mice as an immunodeficient animal was selected for in vivo experiments. All surgeries were performed by the same surgeon. Forty-five nude mice (6 weeks old, weighting 20-25 g) were divided into the PIECs/BAM hydrogel (n=15), PIECs/Col hydrogel (n=15) and unloaded BAM hydrogel (n=15) groups. Subcutaneous incisions were made on the back of each mouse following general anesthesia with intro-abdominal injection of 4% chloral hydrate (0.1 ml/10 g). The PIEC loaded hydrogels (BAM 6 mg/ml or collagen 2.5 mg/ml and PIECs at 1.5×105 cell/ml) measuring 0.4 cm (length) × 0.4 cm (width) × 0.3 cm (height) were implanted subcutaneously in the respective groups, and the incisions were sutured. 2.11 Macroscopic and histological analyses in vivo After 1, 2 and 3 weeks of operation, the nude mice were euthanized, and the hydrogel implants were removed and stained with HE and Masson’s trichome to assess their appearance and degradation. Immunofluorescence staining was also performed using monoclonal antibodies against vascular endothelium (CD31, Boster, Wuhan, China). Positive staining was quantified using Image-Pro Plus 5.1 software (Media Cybernetics, Inc., MD, USA) in 10 different areas for each sample. 2.12 Statistical analysis



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Experimental data were expressed as means ± standard deviation (SD). One-way ANOVA with a post-hoc Tukey test and t-tests were used for comparison via SPSS statistical software (SPSS), and statistical significance was accepted at p < 0.05.

3 RESULTS 3.1 Evaluation of decellularization After decellularization, the color of the fresh bladder changed from pale pink (Fig. 1A) to milky white (Fig. 1B). The effects of decellularization was also verified histologically by H&E and Masson staining. The former showed intact cells with clearly visible nuclei in the untreated porcine bladder (Fig. 1C), which were completely disrupted following decellularization (Fig. 1D). Masson staining showed large collagen fibers (blue) and some muscle fibers (red) in the intact bladder (Fig. 1E), which were not significantly affected by decellularization (Fig. 1F).

Fig. 1 Macroscopic appearance of intact porcine bladder (A) and decellularized BAM sheet (B). H&E 10 ACS Paragon Plus Environment

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staining (C and D) and Masson staining (E and F, black arrow indicates cell nuclei) of intact porcine bladder (C and E) and decellularized BAM sheets (D and F). 3.2 Analysis of hydrogel morphology To determine the proliferative potential of the BAM hydrogels, those with pore sizes similar to collagen hydrogel (2.5 mg/ml) were selected. SEM images showed that while both BAM hydrogels (Fig. 2A-C) and collagen hydrogel (Fig. 2D) had large pore sizes, that of the BAM hydrogels decreased with increasing concentration. The pore sizes of 4 mg/ml, 6 mg/ml and 8 mg/ml BAM hydrogels were 151.2±20.9 m, 120.8±7.1 m and 88.0±10.3 m respectively. The pore size of 2.5 mg/ml collagen hydrogel was 130.0±10.7 m (Fig. 2E), which was similar that of the 6 mg/ml BAM hydrogel and significantly different with the rest (p < 0.05).

Fig. 2 SEM images of 4 mg/ml (A), 6 mg/ml (B) and 8 mg/ml (C) BAM hydrogels, and of 2.5 mg/ml collagen hydrogel (D). The pore size distribution of BAM hydrogels decreased with increasing



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concentrations (E). (Col-2.5 – 2.5 mg/ml collagen hydrogel, BAM-4, BAM-6 and BAM-8 – 4, 6 and 8 mg/ml BAM hydrogels respectively. * p < 0.05). 3.3 Hydrogel turbidity gelation kinetics The gelation of all pre-gel solutions was observed by monitoring the change in the absorbance at 405 nm (Fig. 3A). NA curves were first plotted to compare the gelation of different hydrogels and extrapolate the parameters of gelation kinetics. Hydrogel formation was observed after a lag period, and their turbidity gelation kinetic curves (Fig. 3A and B) showed similar sigmoidal shape which revealed the degree of gelation and optimization of the hydrogels. Collagen hydrogel had the minimal absorbance, while that of the BAM hydrogels increased with their concentration. In addition, the gelation speed (S) was also directly proportional to the concentration of BAM hydrogels (Fig. 3C). The gelation speed of Col-2.5 was significantly faster than that of BAM-4, slower than that of BAM-8, and similar to that of BAM-6. Neither t lag nor t 1/2 showed any significant correlation with BAM concentration (Fig 3D and E). The t lag of BAM-4 was significantly longer than that of BAM-8 and Col-2.5, and there were no significant differences among the other samples (Fig. 3D). The t

1/2

of BAM-4 was also longer than the other three

hydrogel samples, while no significant differences were seen among BAM-6, BAM-8 and Col-2.5 (Fig. 3E).


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Fig. 3 Turbidity gelation kinetics of hydrogels. (A) Turbidity gelation kinetics was monitored by measuring absorbance at 405 nm at 37 °C for 50 min. (B) The normalized absorbance (NA) curve. Data represent means ± standard deviation for n=3. (C) Speed of the turbidity gelation kinetics. (D) Lag time (t lag)

of BAM and collagen hydrogels. (E) 50% gelation time (t

1/2)

of hydrogels. (Col-2.5 – 2.5 mg/ml

collagen hydrogel, BAM-4, BAM-6 and BAM-8 – 4, 6 and 8 mg/ml BAM hydrogels respectively. * p < 0.05). 3.4 Hydrogel rheology analysis and comparison The rheological properties of BAM hydrogels and collagen hydrogel are summarized in Figure 4. Time sweep (Fig. 4A) was used to verify hydrogel formation and optimization of structure. The G’ and G’’ of all hydrogels gradually increased till they reached a maximum equilibrium value, and the solid-like behavior was verified since the G’ was greater than G’’ throughout the testing process. The maximum G’ of BAM-4, BAM-6, BAM-8 and Col-2.5 were 406.1±71.9 Pa, 308.2±40.3 Pa, 76.7±19.8 Pa and 40.5±14.1 Pa respectively (Fig. 4B). The maximum G’ of all BAM hydrogels was thus greater than that 13 ACS Paragon Plus Environment


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of collagen hydrogel, and improved with increasing BAM concentration. Following time sweep, an oscillatory frequency sweep (frequency from 0.1 to 100 rad/s, 1% strain) and an amplitude sweep (strain covering the range 0.1–100%, 1 rad/s frequency) were further performed. The frequency sweep (Fig. 4C) showed that the G’ of hydrogels gradually increased and were significantly larger than G’’ at lower frequencies, indicating that the hydrogel structure was intact. In contrast, G’ dropped sharply and G’’ increased at higher frequencies probably due to the gradual damage of the gel structure. BAM-4 and Col-2.5 may be liquefied with increasing frequency, as indicated by the dramatic decrease in G’ which finally attained a lower value than G’’. The G’ of BAM-6 and BAM-8 were always greater than their respective G’’, indicating better stiffness and mechanical strength of these two hydrogels. Amplitude sweep (Fig. 4D) showed that G’ increased slowly at lower strain ranges and then decreased dramatically. The strain hardening behavior of BAM hydrogels was more obvious than that of the collagen hydrogel, and increased with BAM concentration. Taken together, all rheological measurements of BAM hydrogels showed that high concentrations were associated have higher G’ and better strength, and all BAM hydrogels had better mechanical strength than the collagen hydrogel. Compression tests were used to further characterize and compare the mechanical properties of hydrogels. Col-2.5 could not be tested due to its weak and soft consistency. Broken strength (Fig. 4E) and the maximum compressive moduli (Fig. 4F) of BAM hydrogels increased with their concentrations. The broken strength of BAM-8 was significantly higher than that of the BAM-4 (0.67±0.11 KPa vs 0.44±0.10 KPa), and similar to that of BAM-6 (0.53±0.05 KPa) hydrogel (Fig. 5E). Similarly, compressive modulus of BAM-8 was significantly higher than that of BAM-4 (2.44±0.41 KPa vs 1.60±0.12 KPa), and similar to that of BAM-6 (1.98±0.12 KPa) hydrogel (Fig. 5F).


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Fig. 4 Rheological and compressive properties of hydrogels. (A) Time sweep curves, (B) maximum G’ after complete gelation, (C) frequency sweep (frequency from 0.1 to 100 rad/s, 1% strain), (D) amplitude sweep (strain covering the range 0.1–100%, 1 rad/s frequency), (E) broken strength, and (F) maximum compressive modulus of Col-2.5, BAM-4, BAM-6 and BAM-8 hydrogels. (Broken strength is defined as the compressive strength at hydrogel failure. * p < 0.05). 3.5 Assessment of cell growth in PIEC/hydrogel scaffolds



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Fig. 5 PIECs growth and viability inside the BAM-6 and Col-2.5 hydrogels. (A) Compressed z-stack confocal microscopy images and (B) 3D-cube reconstruction images of PIECs/hydrogel scaffolds by Live/Dead staining (Green= viable cells, Red= dead cells) on day 7. White arrows and white circle indicate the dead cells. (C) H&E staining of PIECs/hydrogel scaffolds (the slices of maximum section and cross section were randomly selected). Red arrow indicates cell nuclei. To monitor the survival of cells inside BAM and collagen hydrogels, PIECs were encapsulated in the hydrogels with similar inner pore size, and stained by a Live/Dead viability assay kit after 7 days. Compressed z-stack fluorescence images (Fig. 5A) showed considerable cell death inside the collagen hydrogel on day 7, whereas the PIECs adequately proliferated for 7 days inside the BAM hydrogels with only negligible cell death. The 3D-cube reconstructed images showed that the PIECs grew throughout the hydrogels, although more dead cells were observed among those growing in Col-2.5 rather than in BAM-6 hydrogel (Fig. 5B). 16 ACS Paragon Plus Environment

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Finally, H&E staining showed intact cells and mature nuclei in random maximum and cross sections of both cell/hydrogel scaffolds. However, the BAM hydrogel had a greater number of nuclei in both sections compared to the collagen hydrogel (Fig. 5C). 3.6 Assessment of cellular morphology

Fig. 6 Morphology of PIECs inside the BAM-6 and Col-2.5 hydrogels on day 7 of culture. Cells were stained with (A) calcein AM to differentiate the viable ones, and with (B) phalloidin and DAPI which respectively dyed the cytoplasm (red) and nuclei (blue). The images in the first panels of (A) and (B) are compressed z-stack images, and those in the second panels are 3D-cube reconstruction images. LOP indicates that the length of 7-day old PIECs inside hydrogels after the 7 day culture. * p < 0.05. PIECs were separately stained with calcein AM and the mixture of phalloidin and DAPI to further evaluate and compare PIEC growth inside hydrogel scaffolds on day 7 of culture. As shown in the compressed z-stack and 3D-cube reconstruction images (Fig. 6A and B), PIECs exhibited an obvious fusiform shape with an average length of 52.1±9.6 m in the BAM-6 hydrogel, and small spherical forms of average length 27.9±2.9 m in the collagen hydrogel (Fig. 6B; p < 0.05). Therefore, PIECs growth was better in the BAM hydrogel compared to the collagen hydrogel.



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3.7 Macroscopic and histological evaluation of hydrogel implant sites All nude mice that were subcutaneously implanted with the PIEC loaded hydrogels survived the duration of the study with no adverse events. The hydrogels were covered with connective tissue, and superficial vessels could be observed with the naked eye (Fig.7A). After 3 weeks of implantation, more blood vessels indicating greater neovascularization were seen on the surface of PIECs/BAM hydrogel compared to the PIECs/collagen and unloaded BAM hydrogels (Fig.7A). Furthermore, the hydrogels gradually degraded during the 3 weeks, with a visible decrease in volume in the 3rd week (Fig.7A). H&E and Masson staining also revealed neovascularization inside the implanted hydrogels at 2 and 3 weeks in each group. During the 1st week post-implantation, there was no obvious neovascularization inside any of the hydrogels, although a few nascent vessels were observed at the junction of the PIECs/BAM hydrogels and native tissue. Compared to the PIECs/Col and unloaded BAM hydrogels, the PIECs/BAM hydrogel interface showed a visible increase in vascular density after 2 and 3 weeks, and the neo-vessels became more enlarged and mature at 3 weeks (Fig. 7B). In addition, an acute inflammatory reaction characterized by segmented neutrophil infiltration was seen surrounding the hydrogels after 1 week of implantation. At weeks 2 and 3, the cell infiltrates in each group were largely mononuclear indicating mild chronic inflammation (Fig. 7B). The structural integrity of the hydrogels was examined by Masson staining, which dyes the collagen blue. The hydrogels showed a marked decrease in the blue-dyed area from 1 to 3 weeks in each group, indicating gradual collagen degradation.


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Fig. 7 Macroscopic and histological analyses of implant sites with PIECs/BAM hydrogel, PIECs/collagen and unloaded BAM hydrogels. (A) Representative pictures showing macroscopic changes at the implant sites at weeks 1, 2 and 3 after implantation in the three groups. White arrows indicate the hydrogel samples. (B) Representative images of H&E and Masson trichrome staining of the implant site tissue at weeks 1, 2 and 3 after implantation in the three groups. The red arrows indicate segmented neutrophils and the black arrow indicates mononuclear cells. The blue area in Masson's trichrome stained sections show the collagen composition of hydrogel scaffolds.


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Fig. 8 Angiogenesis induced by the hydrogels. (A) Immunofluorescence staining of CD31 to assess subcutaneous angiogenesis at weeks 1, 2, and 3 after implantation in the three groups (PIECs/BAM, PIECs/Col, and unloaded BAM). Cell nuclei were counterstained by DAPI. Red arrows indicate vessels. (B) Image analysis for vessel density at each time point. * p < 0.05. To further assess the angiogenic potential of the implanted hydrogels, the CD31 marker expression was detected in the tissue sections (Fig. 8A). The density of CD31+ vessels increased, and the neovessels became enlarged and mature in each group with time. The most significant neovascularization was observed in the PIECs/BAM group compared to the PIECs/Col and unloaded BAM groups (Fig. 8A), with no obvious differences between the latter two at any time point. Furthermore, the neovascularization density of the PIECs/BAM group were significantly higher than the PIECs/Col group (Fig. 8B; p < 0.05), while no obvious differences were seen between the PIECs/Col and unloaded BAM groups (Fig. 8B, p> 0.05).

4 DISCUSSION Thermosensitive ECM hydrogels are a viable option for therapeutic applications due to easy in situ incorporation and simple delivery, ability to be directly injected into even irregularly shaped injury sites, and robust biological activity.40 In this study, BAM was enzymatically solubilized and then induced to form hydrogels of varying concentrations. The inner structure, gelation kinetics and mechanical properties of the hydrogels were characterized, along with their in vitro and in vivo angiogenic potential with compounding PIECs. We found that the pore size of the BAM hydrogels gradually decreased, and their mechanical strength and gelation speed increased with their concentration. In addition, BAM hydrogel loaded with PIECs improved in vitro endothelial cell growth and in vivo vascularization. Adequate cell removal from the source tissues is essential for the in vivo remodeling of the ECM scaffold



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due to the deleterious effects of residual cellular contents.41-43 Furthermore, decellularization also minimizes the immune response in clinical applications. We evaluated the extent of decellularization by H&E and Masson staining, and found similarities with ECM biomaterials from which all nuclei are removed.38,

44-46

The decellularization process did not have a significant effect on collagen fibers,

therefore minimizing the inflammatory response triggered by the hydrogel scaffolds at 2-3 weeks after surgical implantation. Tissue injury generally results in an acute inflammatory reaction in the early stages, during which inflammatory cells including neutrophils and macrophages infiltrate the injury site to clear out dead cells, bacteria and debris, and is critical to tissue healing.47 Therefore, the severe acute inflammatory reaction that we observed during the 1st week after implantation may be due to acute surgical injury rather than the hydrogel itself. We observed the morphologies of BAM and collagen hydrogel by SEM, and compared the pore size. It was found that the pore size of BAM hydrogels gradually decreased with increasing concentration, and BAM-6 had a similar pore size as Col-2.5. The gelation mechanism of BAM hydrogel is not clear. According to the similar pore size (Fig. 2E), similar speed of the turbidity gelation kinetics (Fig. 3C) of BAM-6 and Col-2.5, we therefore hypothesized that the GAGs and other ECM proteins inside BAM may deteriorate the BAM gelation behavior and result in the similar pore size of BAM-6 with Col-2.5. Turbidimetric gelation kinetics and rheology are the primary methods used to assess the viscoelastic properties of ECM hydrogels.48 We therefore assessed the turbidimetric gelation kinetics, rheology, and compression of BAM hydrogels to determine their kinetic and mechanical properties. Both BAM and collagen hydrogels showed sigmoidal gelation curves similar to that seen in other ECM based hydrogels.25,

36-37, 39

indicating gelation. ECM hydrogel formation is a collagen-based self-assembly as

previously reported.49 Furthermore, the time sweep of rheological analysis revealed that the optimization


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and gelation process of BAM hydrogel was similar to ECM hydrogels of lung,50 bone38 and heart51 origin, in addition to a positive correlation between G’ and concentration. The maximum equilibrium value of G’ was related to stiffness, and solid-like behavior of the hydrogel was confirmed when the G’ was considerably greater than the G’’.36,

48

The frequency sweep results indicated that the G’ of BAM

hydrogels were frequency dependent and decreased at higher frequencies. The strain sweep analysis showed different strain behavior of BAM hydrogel compared to the other ECM hydrogels.38,

50

Both

rheological and compression analyses of the BAM hydrogels indicated that their mechanical strength increased with concentration, as reported for other ECM hydrogels.25, 37, 39 Therefore, the rheological G’ and compressive strength of BAM hydrogels can be manipulated to a certain extent by varying the ECM concentration. Multiple scaffold design parameters influence the transport of nutrients and metabolic waste products, with pore size being a key factor of vascularization. Large pore sizes can enhance the nutrient transfer to maintain and improve the viability of cells, and contribute to ECs proliferation, infiltration and differentiation.52 Theoretically, this indicates similar vascular EC infiltration into the hydrogels, as well as proliferation and differentiation. Therefore, to avoid any negative effect of pore size on potential vascularization and also sustain reasonable mechanical strength, BAM-6 was selected to compare its angiogenic potential with that of the collagen hydrogel control, after PIECs loading. The pre-vascularization potential of BAM hydrogel with compounding PIECs was characterized by in vitro cell three-dimensional culture. After 7 days of in vitro culture, adequate proliferation of the PIECs was seen throughout the BAM hydrogels, while more dead cells were observed in the collagen hydrogel. This indicated that the BAM hydrogels provided a more conducive microenvironment for PIECs growth, most likely due to the release of inherent growth factors. Compared to an individual ECM component in



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the collagen hydrogel, BAM contains at least ten different growth factors such as VEGF, PDGF-BB and KGF,36, 53 making it more bioactive. Recently, the in vivo angiogenic potential of ECM hydrogel derived from small intestinal submucosa (SIS) was associated with the release of VEGF and other inherent growth factors.54 In addition, our previous study showed that these endogenous growth factors were well-preserved in the BAM hydrogel.26 As target receptors on ECs, VEGF is a key player in both physiological and pathological angiogenesis.55-57 Therefore, on account of these growth factors, BAM hydrogel resulted in more viable ECs compared to collagen hydrogel in vitro. Successful tissue-engineering depends on the outcome of a number of complex processes, of which rapid vascularization of the implanted scaffold is critical. To evaluate the angiogenic potential of BAM hydrogel in vivo, PIECs/BAM, PIECs/Col and unloaded BAM hydrogel scaffolds were subcutaneously implanted in nude mice. Studies have reported the utility of compounding ECs in constructing models of angiogenesis in hydrogels,28,

58

Furthermore, our previous study showed that the maximum amount of

VEGF and PDGF-BB released in the BAM hydrogels were >100 times and 2 times respectively compared with that in the BAM sheets.26 Based on this coordinated inter play of the ECs and growth factors, PIECs/BAM hydrogel is the most obvious choice for angiogenesis compared to the other tested hydrogels.

5 CONCLUSION The similar pore sizes of 6 mg/ml BAM and 2.5 mg/ml collagen hydrogels was the basis of comparing their angiogenesis potential. Due to the inherent growth factors, BAM hydrogel promoted PIECs growth better than collagen hydrogel in vitro, and the coordinated interplay of ECs and these growth factors contributed to in vivo angiogenesis. Our findings provide the experimental basis for using BAM hydrogel scaffolds in organ repair, reconstruction and vascularization. The BAM hydrogel with inherent bioactive 24 ACS Paragon Plus Environment

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factors may be further used as a delivery system for tissue engineering via injection or coating in animal or clinic application.

Author Contributions Yaopeng Zhang designed the project and revised the manuscript. Wenjing Liu performed most of the experiments except animal experiments, and wrote the manuscript. Jianwen Huang directed Nailong Cao to perform the in vivo characterization. Suna Fan, Huihui Zhang, Huili Shao, Chengbo Cao, and Lujie Song provided advices to gel preparation, rheology, in vitro study and in vivo experiment, respectively.

Disclosure of potential conflict of interest We declare that there are no conflicts of interest related to this work.

ORCID Yaopeng Zhang: 0000-0002-7175-6150

Acknowledgements This work is sponsored by the National Key Research and Development Program of China (2018YFC1105802, 2016YFA0201702), National Natural Science Foundation of China (21674018, 81600524), National Key Research and Development Program of China (2018YFC1106002), International Joint Laboratory for Advanced fiber and Low-dimension Materials (18520750400), “Shuguang Program” supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (15SG30), and State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University (LK1408, KF1816).



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References 1. Spinali, K. L.; Schmuck, E. G., Natural Sources of Extracellular Matrix for Cardiac Repair. Adv Exp Med Biol. 2018, 1098, 115-130. 2. D'Amore, A.; Yoshizumi, T.; Luketich, S. K.; Wolf, M. T.; Gu, X. Z.; Cammarata, M.; Hoff, R.; Badylak, S. F.; Wagner, W. R., Bi-layered Polyurethane - Extracellular Matrix Cardiac Patch Improves Ischemic Ventricular Wall Remodeling in a Rat Model. Biomaterials. 2016, 107, 1-14. 3. Gubareva, E. A.; Sjoqvist, S.; Gilevich, I. V.; Sotnichenko, A. S.; Kuevda, E. V.; Lim, M. L.; Feliu, N.; Lemon, G.; Danilenko, K. A.; Nakokhov, R. Z.; Gumenyuk, I. S.; Grigoriev, T. E.; Krasheninnikov, S. V.; Pokhotko, A. G.; Basov, A. A.; Dzhimak, S. S.; Gustafsson, Y.; Bautista, G.; Rodriguez, A. B.; Pokrovsky, V. M.; Jungebluth, P.; Chvalun, S. N.; Holterman, M. J.; Taylor, D. A.; Macchiarini, P., Orthotopic Transplantation of a Tissue Engineered Diaphragm in Rats. Biomaterials. 2016, 77, 320-335. 4. Wassenaar, J. W.; Boss, G. R.; Christman, K. L., Decellularized Skeletal Muscle as an in Vitro Model for Studying Drug-extracellular Matrix Interactions. Biomaterials. 2015, 64, 108-114. 5. Valentin, J. E.; Turner, N. J.; Gilbert, T. W.; Badylak, S. F., Functional Skeletal Muscle Formation with a Biologic Scaffold. Biomaterials. 2010, 31 (29), 7475-7484. 6. Badylak, S. F.; Freytes, D. O.; Gilbert, T. W., Extracellular Matrix as a Biological Scaffold Material: Structure and Function. Acta. Biomater. 2009, 5 (1), 1-13. 7. Beattie, A. J.; Gilbert, T. W.; Guyot, J. P.; Yates, A. J.; Badylak, S. F., Chemoattraction of Progenitor Cells by Remodeling Extracellular Matrix Scaffolds. Tissue. Eng. Part. A. 2009, 15 (5), 1119-1125. 8. Rangel-Argote, M.; Claudio-Rizo, J. A.; Mata-Mata, J. L.; Mendoza-Novelo, B., Characteristics of Collagen-Rich Extracellular Matrix Hydrogels and Their Functionalization with Poly(ethylene glycol) Derivatives for Enhanced Biomedical Applications: A Review. ACS Appl Bio Mater. 2018, 1 (5), 1215-1228. 9. Kaphle, P.; Li, Y.; Yao, L., The Mechanical and Pharmacological Regulation of Glioblastoma Cell Migration in 3D Matrices. J CellPhysiol. 2019, 234 (4), 3948-3960.. 10. Vorotnikova, E.; McIntosh, D.; Dewilde, A.; Zhang, J.; Reing, J. E.; Zhang, L.; Cordero, K.; Bedelbaeva, K.; Gourevitch, D.; Heber-Katz, E.; Badylak, S. F.; Braunhut, S. J., Extracellular Matrix-derived Products Modulate Endothelial and Progenitor Cell Migration and Proliferation in Vitro and Stimulate Regenerative Healing in Vivo. Matrix. Biol. 2010, 29 (8), 690-700. 11. Valentin, J. E.; Badylak, J. S.; Mccabe, G. P.; Badylak, S. F., Extracellular Matrix Bioscaffolds for Orthopaedic Applications - A Comparative Histologic Study. J. Bone. Joint. Surg. Am. 2006, 88a (12), 2673-2686. 12. Hoganson, D. M.; O'Doherty, E. M.; Owens, G. E.; Harilal, D. O.; Goldman, S. M.; Bowley, C. M.; Neville, C. M.; Kronengold, R. T.; Vacanti, J. P., The Retention of Extracellular Matrix Proteins and Angiogenic and Mitogenic Cytokines in a Decellularized Porcine Dermis. Biomaterials. 2010, 31 (26), 6730-6737. 13. Badylak, S. E., The Extracellular Matrix as a Scaffold for Tissue Reconstruction. Semin. Cell. Dev. Biol. 2002, 13 (5), 377-383. 14. El Kassaby, A.; AbouShwareb, T.; Atala, A., Randomized Comparative Study Between Buccal Mucosal and Acellular Bladder Matrix Grafts in Complex Anterior Urethral Strictures. J. Urol. 2008, 179 (4), 1432-1436. 15. Li, C.; Xu, Y. M.; Liu, Z. S.; Li, H. B., Urethral Reconstruction With Tissue Engineering and RNA Interference Techniques in Rabbits. Urology. 2013, 81 (5), 1075-1080. 26 ACS Paragon Plus Environment

Page 27 of 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


16. Atala, A., Bioengineered Tissues for Urogenital Repair in Children. Pediatr. Res. 2008, 63 (5), 569-575. 17. Kassaby, A. W. E.; Aboushwareb, T.; Atala, A., Randomized Comparative Study Between Buccal Mucosal and Acellular Bladder Matrix Grafts in Complex Anterior Urethral Strictures. J. Urol. 2008, 179 (4), 1432-1436. 18. Li, H.; Xu, Y.; Xie, H.; Li, C.; Song, L.; Feng, C.; Zhang, Q.; Xie, M.; Wang, Y.; Lv, X., Epithelial-differentiated Adipose-derived Stem Cells Seeded Bladder Acellular Matrix Grafts for Urethral Reconstruction: An Animal Model. Tissue. Eng. Part. A. 2014, 20 (3-4), 774-784. 19. Huang, J. W.; Xie, M. K.; Zhang, Y.; Wei, G. J.; Li, X.; Li, H. B.; Wang, J. H.; Zhu, W. D.; Li, C.; Xu, Y. M.; Song, L. J., Reconstruction of Penile Urethra with the 3-dimensional Porous Bladder Acellular Matrix in a Rabbit Model. Urology. 2014, 84 (6), 1499-1505. 20. Li, C.; Xu, Y. M.; Song, L. J.; Fu, Q.; Cui, L.; Yin, S., Urethral Reconstruction Using Oral Keratinocyte Seeded Bladder Acellular Matrix Grafts. J. Urol. 2008, 180 (4), 1538-1542. 21. Cabral, J.; Moratti, S. C., Hydrogels for biomedical applications. Future. Med. Chem. 2011, 3 (15), 1877-1888. 22. Spang, M. T.; Christman, K. L., Extracellular Matrix Hydrogel Therapies: In Vivo Applications and Development. Acta. Biomater. 2018, 68, 1-14. 23. Morris, A. H.; Lee, H.; Xing, H.; Stamer, D. K.; Tan, M.; Kyriakides, T. R., Tunable Hydrogels Derived from Genetically Engineered Extracellular Matrix Accelerate Diabetic Wound Healing. ACS Appl Mater Interfaces. 2018, 10 (49), 41892-41901. 24. Sonnenberg, S. B.; Rane, A. A.; Liu, C. J.; Rao, N.; Agmon, G.; Suarez, S.; Wang, R.; Munoz, A.; Bajaj, V.; Zhang, S.; Braden, R.; Schup-Magoffin, P. J.; Kwan, O. L.; DeMaria, A. N.; Cochran, J. R.; Christman, K. L., Delivery of an Engineered HGF Fragment in an Extracellular Matrix-derived Hydrogel Prevents Negative LV Remodeling Post-myocardial Infarction. Biomaterials. 2015, 45, 56-63. 25. Wolf, M. T.; Daly, K. A.; Brennan-Pierce, E. P.; Johnson, S. A.; Carruthers, C. A.; D'Amore, A.; Nagarkar, S. R.; Velankar, S. S.; Badylak, S. F., A Hydrogel Derived from Decellularized Dermal Extracellular Matrix. Biomaterials. 2012, 33 (29), 7028-7038. 26. Jiang, D.; Huang, J. W.; Shao, H. L.; Hu, X. C.; Song, L. J.; Zhang, Y. P., Characterization of Bladder Acellular Matrix Hydrogel with Inherent Bioactive Factors. Mat. Sci. Eng. C. 2017, 77, 184-189. 27. Seif-Naraghi, S. B.; Horn, D.; Schup-Magoffin, P. J.; Christman, K. L., Injectable Extracellular Matrix Derived Hydrogel Provides a Platform for Enhanced Retention and Delivery of a Heparin-Binding Growth Factor. Acta. Biomater. 2012, 8 (10), 3695-3703. 28. Morin, K. T.; Tranquillo, R. T., In Vitro Models of Angiogenesis and Vasculogenesis in Fibrin Gel. Exp. Cell. Res. 2013, 319 (16), 2409-2417. 29. Morin, K. T.; Tranquillo, R. T., Guided Sprouting from Endothelial Spheroids in Fibrin Gels Aligned by Magnetic Fields and Cell-induced Gel Compaction. Biomaterials. 2011, 32 (26), 6111-6118. 30. Qian, Y. F.; Zhang, K. H.; Chen, F.; Ke, Q. F.; Mo, X. M., Cross-Linking of Gelatin and Chitosan Complex Nanofibers for Tissue-Engineering Scaffolds. J Biomat Sci-Polym E. 2011, 22 (8), 1099-1113. 31. Nissen, L. J.; Cao, R.; Hedlund, E.-M.; Wang, Z.; Zhao, X.; Wetterskog, D.; Funa, K.; Bråkenhielm, E.; Cao, Y., Angiogenic Factors FGF2 and PDGF-BB Synergistically Promote Murine Tumor Neovascularization and Metastasis. J. Clin. Invest. 2007, 117 (10), 2766-2777. 32. Nourse, M. B.; Halpin, D. E.; Scatena, M.; Mortisen, D. J.; Tulloch, N. L.; Hauch, K. D.; Torok-Storb, B.; Ratner, B. D.; Pabon, L.; Murry, C. E., VEGF Induces Differentiation of Functional Endothelium from Human Embryonic Stem Cells: Implications for Tissue Tngineering. Arterioscler. Thromb. Vasc. Biol. 2010, 30 (1), 80-89. 27 ACS Paragon Plus Environment


Page 28 of 30

33. Huang, J. W.; Xu, Y. M.; Li, Z. B.; Murphy, S. V.; Zhao, W. X.; Liu, Q. Q.; Zhu, W. D.; Fu, Q.; Zhang, Y. P.; Song, L. J., Tissue Performance of Bladder Following Stretched Electrospun Silk Fibroin Matrix and Bladder Acellular Matrix Implantation in a Rabbit Model. J. Biomed. Mater. Res. A. 2016, 104 (1), 9-16. 34. Li, H. B.; Xu, Y. M.; Xie, H.; Li, C.; Song, L. J.; Feng, C.; Zhang, Q.; Xie, M. K.; Wang, Y.; Lv, X. G., Epithelial-Differentiated Adipose-Derived Stem Cells Seeded Bladder Acellular Matrix Grafts for Urethral Reconstruction: An Animal Model. Tissue. Eng. Part. A. 2014, 20 (3-4), 774-784. 35. Cao, N. L.; Song, L. J.; Liu, W. J.; Fan, S. N.; Jiang, D.; Mu, J. G.; Gu, B. J.; Xu, Y. M.; Zhang, Y. P.; Huang, J. W., Prevascularized Bladder Acellular Matrix Hydrogel/Silk Fibroin Composite Scaffolds Promote the Regeneration of Urethra in a Rabbit Model. Biomed Mater. 2019, 14 (1), 015002-015002. 36. Freytes, D. O.; Martin, J.; Velankar, S. S.; Lee, A. S.; Badylak, S. F., Preparation and Rheological Characterization of a Gel Form of the Torcine Urinary Bladder Matrix. Biomaterials. 2008, 29 (11), 1630-1637. 37. Wu, J. L.; Ding, Q.; Dutta, A.; Wang, Y. Z.; Huang, Y. H.; Weng, H.; Tang, L. P.; Hong, Y., An Injectable Extracellular Matrix Derived Hydrogel for Meniscus Repair and Regeneration. Acta. Biomater. 2015, 16, 49-59. 38. Sawkins, M. J.; Bowen, W.; Dhadda, P.; Markides, H.; Sidney, L. E.; Taylor, A. J.; Rose, F. R. A. J.; Badylak, S. F.; Shakeshehh, K. M.; White, L. J., Hydrogels Derived from Demineralized and Decellularized Bone Extracellular Matrix. Acta. Biomater. 2013, 9 (8), 7865-7873. 39. Medberry, C. J.; Crapo, P. M.; Siu, B. F.; Carruthers, C. A.; Wolf, M. T.; Nagarkar, S. P.; Agrawal, V.; Jones, K. E.; Kelly, J.; Johnson, S. A.; Velankar, S. S.; Watkins, S. C.; Modo, M.; Badylak, S. F., Hydrogels Derived from Central Nervous System Extracellular Matrix. Biomaterials. 2013, 34 (4), 1033-1040. 40. Domb, A.; Mikos, A. G., Matrices and Scaffolds for Drug Delivery in Tissue Engineering. Adv. Drug. Deliver. Rev. 2007, 59 (4), 185-186. 41. Brown, B. N.; Valentin, J. E.; Stewart-Akers, A. M.; McCabe, G. P.; Badylak, S. F., Macrophage Phenotype and Remodeling Outcomes in Response to Biologic Scaffolds With and Without a Cellular Component. Biomaterials. 2009, 30 (8), 1482-1491. 42. Crapo, P. M.; Gilbert, T. W.; Badylak, S. F., An Overview of Tissue and Whole Organ Decellularization Processes. Biomaterials. 2011, 32 (12), 3233-3243. 43. Keane, T. J.; Londono, R.; Turner, N. J.; Badylak, S. F., Consequences of Ineffective Decellularization of Biologic Scaffolds on the Host Response. Biomaterials. 2012, 33 (6), 1771-1781. 44. Prest, T. A.; Yeager, E.; LoPresti, S. T.; Zygelyte, E.; Martin, M. J.; Dong, L. Y.; Gibson, A.; Olutoye, O. O.; Brown, B. N.; Cheetham, J., Nerve-specific, Xenogeneic Extracellular Matrix Hydrogel Promotes Recovery Following Peripheral Nerve Injury. J. Biomed. Mater. Res. A. 2018, 106 (2), 450-459. 45. Ryzhuk, V.; Zeng, X. X.; Wang, X. J.; Melnychuk, V.; Lankford, L.; Farmer, D.; Wang, A. J., Human Amnion Extracellular Matrix Derived Bioactive Hydrogel for Cell Delivery and Tissue Engineering. Mat. Sci. Eng. C. 2018, 85, 191-202. 46. Fu, Y.; Fan, X.; Tian, C.; Luo, J.; Zhang, Y.; Deng, L.; Qin, T.; Lv, Q., Decellularization of Porcine Skeletal Muscle Extracellular Matrix for the Formulation of a Matrix Hydrogel: a Preliminary Study. J. Cell. Mol. Med. 2016, 20 (4), 740-749. 47. Lei, C.; Qi, X.; Zhai, Q.; Tahtinen, M.; Fei, Z.; Chen, L.; Xu, Y.; Qi, S.; Feng, Z., Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Repair. Theranostics. 2017, 7 (1), 117-131. 48. Saldin, L. T.; Cramer, M. C.; Velankar, S. S.; White, L. J.; Badylak, S. F., Extracellular Matrix 28 ACS Paragon Plus Environment

Page 29 of 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


Hydrogels from Decellularized Tissues: Structure and Function. Acta. Biomater. 2017, 49, 1-15. 49. Huleihel, L.; Hussey, G. S.; Naranjo, J. D.; Zhang, L.; Dziki, J. L.; Turner, N. J.; Stolz, D. B.; Badylak, S. F., Matrix-bound Nanovesicles within ECM Bioscaffolds. Sci. Adv. 2016, 2 (6), e1600502-e1600502. 50. Pouliot, R. A.; Link, P. A.; Mikhaiel, N. S.; Schneck, M. B.; Valentine, M. S.; Gninzeko, F. J. K.; Herbert, J. A.; Sakagami, M.; Heise, R. L., Development and Characterization of a Naturally Derived Lung Extracellular Matrix Hydrogel. J. Biomed. Mater. Res. A. 2016, 104 (8), 1922-1935. 51. Johnson, T. D.; Lin, S. Y.; Christman, K. L., Tailoring Material Properties of a Nanofibrous Extracellular Matrix Derived Hydrogel. Nanotechnology. 2011, 22 (49), 494015-494015. 52. Ng, K. W.; Khor, H. L.; Hutmacher, D. W., In Vitro Characterization of Natural and Synthetic Dermal Matrices Cultured with Human Dermal Fibroblasts. Biomaterials. 2004, 25 (14), 2807-2818. 53. Chun, S. Y.; Lim, G. J.; Kwon, T. G.; Kwak, E. K.; Kim, B. W.; Atala, A.; Yoo, J. J., Identification and Characterization of Bioactive Factors in Bladder Submucosa Matrix. Biomaterials. 2007, 28 (29), 4251-4256. 54. Wang, W.; Zhang, X.; Chao, N. N.; Qin, T. W.; Ding, W.; Zhang, Y.; Sang, J. W.; Luo, J. C., Preparation and Characterization of Pro-angiogenic Gel Derived from Small Intestinal Submucosa. Acta. Biomater. 2016, 29, 135-148. 55. Carmeliet, P., Mechanisms of Angiogenesis and Arteriogenesis. Nat. Med. 2000, 6 (4), 389-395. 56. Ferrara, N.; DavisSmyth, T., The Biology of Vascular Endothelial Growth Factor. Endocr. Rev. 1997, 18 (1), 4-25. 57. Ferrara, N.; Gerber, H. P.; LeCouter, J., The Biology of VEGF and Its Receptors. Nat. Med. 2003, 9 (6), 669-676. 58. Heller, M.; Frerick-Ochs, E. V.; Bauer, H. K.; Schiegnitz, E.; Flesch, D.; Brieger, J.; Stein, R.; Al-Nawas, B.; Brochhausen, C.; Thüroff, J. W., Tissue Engineered Pre-vascularized Buccal Mucosa Equivalents Utilizing a Primary Triculture of Epithelial Cells, Endothelial Cells and Fibroblasts. Biomaterials. 2015, 77, 207-215.



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Graphical Abstract


Angiogenesis Potential of Bladder Acellular Matrix Hydrogel by

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