Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments

Affiliations

¹ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, School of Engineering, University of Kansas, Lawrence, KS, USA.
² Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, School of Engineering, University of Kansas, Lawrence, KS, USA.
³ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁴ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Cardiovascular Surgery, Heinrich Heine University, Medical Faculty, 40225 Duesseldorf, Germany.
⁵ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Orthopaedic and Trauma Surgery, Pontificia Universidad Católica del Ecuador, Quito-Ecuador.
⁶ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
⁷ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, Doha, Qatar.
⁸ Department of Electrical and Computer Engineering, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
⁹ Center of Nanotechnology, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
¹⁰ Department of Biomedical Engineering and Department of Materials Science and Engineering, Texas A&M University, 5024 Emerging Technology Building, College Station, TX 77843.
¹¹ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Bioindustrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea; Department of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia; WPI-Advanced Institute for Materials Research, Tohoku University, Sendai, Japan.

PMID: 27525102
PMCID: PMC4980085
DOI: 10.1039/C5TB02745D

Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments

Arghya Paul et al. J Mater Chem B. 2016.

. 2016 May 28;4(20):3544-3554.

doi: 10.1039/C5TB02745D. Epub 2016 Feb 4.

Authors

Affiliations

¹ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, School of Engineering, University of Kansas, Lawrence, KS, USA.
² Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, School of Engineering, University of Kansas, Lawrence, KS, USA.
³ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁴ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Cardiovascular Surgery, Heinrich Heine University, Medical Faculty, 40225 Duesseldorf, Germany.
⁵ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Orthopaedic and Trauma Surgery, Pontificia Universidad Católica del Ecuador, Quito-Ecuador.
⁶ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
⁷ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, Doha, Qatar.
⁸ Department of Electrical and Computer Engineering, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
⁹ Center of Nanotechnology, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
¹⁰ Department of Biomedical Engineering and Department of Materials Science and Engineering, Texas A&M University, 5024 Emerging Technology Building, College Station, TX 77843.
¹¹ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Bioindustrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea; Department of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia; WPI-Advanced Institute for Materials Research, Tohoku University, Sendai, Japan.

PMID: 27525102
PMCID: PMC4980085
DOI: 10.1039/C5TB02745D

Abstract

The ability to modulate stem cell differentiation in a three dimensional (3D) microenvironment for bone tissue engineering in absence of exogenous pharmaceutical agents such as bone morphogenic protein (BMP-2) remains a challenge. In this study, we introduce extracellular matrix (ECM)-mimicking nanocomposite hydrogels to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) for bone regeneration in absence of any osteoinducting factors. In particular, we have reinforced photocrosslinkable collagen-based matrix (gelatin methacryloyl, GelMA) used disk-shaped nanosilicates (nSi), a new class of two-dimensional (2D) nanomaterials. We show that nanoengineered hydrogels supported migration and proliferation of encapsulated hMSCs, with no signs of cell apoptosis or inflammatory cytokine responses. The addition of nSi significantly enhances osteogenic differentiation of encapsulated hMSCs as evident by the increase in alkaline phosphates (ALP) activity and deposition of biomineralized matrix compared to GelMA without nSi. We also show that microfabricated nanoengineered microgels can be used to pattern and control cellular behaviour. Furthermore, we also show that nanoengineered hydrogel have high biocompatibility as determined by in vivo experiments using immunocompetent rat model. Specifically, the hydrogels showed minimum localized immune responses, indicating it ability for tissue engineering applications. Overall, we showed the ability of nanoengineered hydrogels loaded with 2D nanosilicates for osteogenic differentiation of stem cells in vitro, in absence of any growth factors such as BMP-2. Our in vivo studies show high biocompatibility of nanocomposites and show the potential for growth factor free bone regeneration.

Keywords: bone regeneration; hydrogels; nanomaterials; nanosilicates; tissue engineering.

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Figures

**Figure 1**
Characterization of photocrosslinked GelMA-nSi hydrogel. **(A)** Schematic of GelMA-nSi hydrogel prepared by UV crosslinking GelMA prepolymer with NS. **(B)** SEM images of cross-sections of GelMA and GelMA-nSi hydrogels (Scale: 100μm). **(C)** Percentage porous area covered was quantified by SEM image analysis of 7% GelMA hydrogels with different concentrations of NS. **(D)** FTIR spectra of GelMA-nSi after UV crosslinking, confirming the presence of silicate and gelatin components in the nanocomposite construct. **(E)** Compressive mechanical properties of 7% GelMA hydrogel with different nSi concentrations with respect to compressive modulus. **(F)** Degradation profiles of 7% GelMA hydrogels with various nSi concentrations when exposed to collagenase for 24h. Data represent Mean ± SD (n=3). *=p< 0.05 compared to control GelMA group (0% nSi).

**Figure 2**
*In vitro* cell behavior of hMSCs encapsulated in GelMA-nSi nanocomposite hydrogel. **(A)** Schematic for photo-encapsulation hMSCs in GelMA-nSi hydrogel consisting of nanosilicates and gelatin matrix. Lower left panel microphotograph represents H&E stained cross-section of 7% GelMA hydrogels with 0.05% nSi with dark blue spots showing encapsulated cell nuclei after a day in culture. Scale bar: 200μm. **(B)** Fluorescence images of F-actin cytoskeleton stained cells (in green) with nuclei (in blue) grown in 3D 7% GelMA hydrogel constructs with different nSi concentrations for 4 days in normal growth media. F-actin cytoskeleton is an indicator and biophysical regulator of cell shape and morphology. F-actin staining confirmed the normal cell architectures in all the experimental groups, except the 0.5% nSi group where proper cell migration and normal cytoskeleton structures were limited. Scale bar: 100μm. **(C)** Metabolic activity of encapsulated cells in 7% GelMA hydrogels with various nSi concentrations was measured by MTS assay after 24h to determine the cytotoxic effects on the formulated hydrogels. **(D)** Caspase 3/7 activity assay was used to quantify the apoptotic effects of different nSi concentrations on encapsulated cells. Data represent Mean ± SD (n=3). *=p< 0.05 & ***=p< 0.001 compared to control GelMA group (0% nSi).

**Figure 3**
Effect of GelMA-nSi hydrogel on differentiation potential of encapsulated hMSCs. **(A)** Schematic of hMSCs differentiated to osteogenic lineage when grown in 3D GelMA-nSi hydrogel environment for 21 days. Quantification of osteogenic differentiation of hMSCs within hydrogels was performed by **(B)** ALP activity, **(C)** alizarin red staining and **(D)** calcium quantification in 7% GelMA hydrogels with varying concentration of NS after Day 21 of culture in osteoconductive media with no supplementary growth factors or drugs. **(E)** Expression of three osteogenic markers - osteocalcin, osteopontin and Runt-related transcription factor 2 (RunX2) by the encapsulated cells were detected by immunostaining and fluorescence microscopy. Osteogenic markers were stained in red, while cell nuclei in blue. **(F)** Confocal laser microscopy image of hMSCs in GelMA-nSi (0.05%) hydrogel expressing osteocalcin, immunostained in red. The picture represents a single z-plane image and x-z and y-z cross-sectional images of the encapsulated cells in hydrogel. Scale bars: 100μm. Data represent Mean ± SD (n=3). *=p< 0.05, **=p< 0.01 & ***=p< 0.001 compared to control GelMA only group (0% nSi).

**Figure 4**
*In vivo* biocompatibility of the GelMA-nSi hydrogels. (A) Hydrogel constructs with different formulations (0%, 0.01%, 0.05%, 0.5% nSi) were implanted subcutaneously in immunocompetent rat model and collected after 3 and 14 days to study the inflammatory responses. (B) Histological results with H&E staining of Ctrl 0% and 0.05% nSi constructs with 7% GelMA showed no signs of inflammation in the adjacent tissue region. * indicates the host tissue regions and white arrows show the tight interface between intact hydrogel and tissues. A magnified image of the hydrogel region confirms there was no infiltration of macrophages or neutrophils into the hydrogel region. This was further confirmed by (C) immunohistochemistry analysis for CD68 and CD3 inflammatory markers in the hydrogel/tissue interface region where **(D)** represents the microphotographs of different groups at day 14 post implantation immunostained with CD68 (in green) and CD3 (in red), nuclei (DAPI, in blue) and corresponding phase contrast images. Scale bar: 100μm. The results demonstrate that the hydrogels remained firmly attached to the tissue surface without invasion by foreign macrophage and T-cells in Ctrl 0%, 0.01% and 0.05% nSi groups. However, 7% GelMA 0.5% nSi group showed marked increase in macrophage levels compared to the other groups. Data represent Mean ± SD (n=3). ***=p< 0.001 compared to control GelMA group (0% nSi).

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References

1. Amini R, Laurencin CT, Nukavarapu SP. Critical reviews in biomedical engineering. 2012;40:363–408. - PMC - PubMed
1. Liu Y, Lim J, Teoh SH. Biotechnology advances. 2013;31:688–705. - PubMed
1. Seong JM, Kim BC, Park JH, Kwon IK, Mantalaris A, Hwang YS. Biomedical materials. 2010;5:062001. - PubMed
1. Guvendiren M, Burdick JA. Nature communications. 2012;3:792. - PubMed
1. Annabi N, Tamayol A, Uquillas JA, Akbari M, Bertassoni LE, Cha C, Camci-Unal G, Dokmeci MR, Peppas NA, Khademhosseini A. Advanced materials. 2014;26:85–123. - PMC - PubMed

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Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments

Affiliations

Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments

Authors

Affiliations

Abstract

Figures

References

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