Dynamic and Cell-Infiltratable Hydrogels as Injectable Carrier of Therapeutic Cells and Drugs for Treating Challenging Bone Defects

doi:10.1021/acscentsci.8b00764

. 2019 Mar 27;5(3):440-450.

doi: 10.1021/acscentsci.8b00764. Epub 2019 Feb 13.

Dynamic and Cell-Infiltratable Hydrogels as Injectable Carrier of Therapeutic Cells and Drugs for Treating Challenging Bone Defects

Qian Feng^{1

2}, Jiankun Xu³, Kunyu Zhang², Hao Yao³, Nianye Zheng³, Lizhen Zheng³, Jiali Wang³, Kongchang Wei^{2

4}, Xiufeng Xiao¹, Ling Qin³, Liming Bian^{2

5

6

7

8}

Affiliations

¹ Fujian Provincial Key Laboratory of Advanced Materials Oriented Chemical Engineering, College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350007, China.
² Department of Biomedical Engineering, The Chinese University of Hong Kong, William M.W. Mong Building, Shatin, Hong Kong SAR, China.
³ Department of Orthopaedic and Traumatology and Innovative Orthopaedic Biomaterial and Drug Translational Research Laboratory of Li Ka Shing Institute of Health, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
⁴ Laboratory for Biomimetic Membranes and Textiles, Empa, Swiss Federal Laboratories for Materials Science and Technology, 9014 St., Gallen, Switzerland.
⁵ Translational Research Centre of Regenerative Medicine and 3D Printing Technologies of Guangzhou Medical University, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China.
⁶ Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518172, China.
⁷ China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou 310058, China.
⁸ Centre for Novel Biomaterials, The Chinese University of Hong Kong, Hong Kong SAR, China.

PMID: 30937371
PMCID: PMC6439455
DOI: 10.1021/acscentsci.8b00764

Dynamic and Cell-Infiltratable Hydrogels as Injectable Carrier of Therapeutic Cells and Drugs for Treating Challenging Bone Defects

Qian Feng et al. ACS Cent Sci. 2019.

. 2019 Mar 27;5(3):440-450.

doi: 10.1021/acscentsci.8b00764. Epub 2019 Feb 13.

Authors

Qian Feng^{1

2}, Jiankun Xu³, Kunyu Zhang², Hao Yao³, Nianye Zheng³, Lizhen Zheng³, Jiali Wang³, Kongchang Wei^{2

4}, Xiufeng Xiao¹, Ling Qin³, Liming Bian^{2

5

6

7

8}

Affiliations

¹ Fujian Provincial Key Laboratory of Advanced Materials Oriented Chemical Engineering, College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350007, China.
² Department of Biomedical Engineering, The Chinese University of Hong Kong, William M.W. Mong Building, Shatin, Hong Kong SAR, China.
³ Department of Orthopaedic and Traumatology and Innovative Orthopaedic Biomaterial and Drug Translational Research Laboratory of Li Ka Shing Institute of Health, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
⁴ Laboratory for Biomimetic Membranes and Textiles, Empa, Swiss Federal Laboratories for Materials Science and Technology, 9014 St., Gallen, Switzerland.
⁵ Translational Research Centre of Regenerative Medicine and 3D Printing Technologies of Guangzhou Medical University, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China.
⁶ Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518172, China.
⁷ China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou 310058, China.
⁸ Centre for Novel Biomaterials, The Chinese University of Hong Kong, Hong Kong SAR, China.

PMID: 30937371
PMCID: PMC6439455
DOI: 10.1021/acscentsci.8b00764

Abstract

Biopolymeric hydrogels have been widely used as carriers of therapeutic cells and drugs for biomedical applications. However, most conventional hydrogels cannot be injected after gelation and do not support the infiltration of cells because of the static nature of their network structure. Here, we develop unique cell-infiltratable and injectable (Ci-I) gelatin hydrogels, which are physically cross-linked by weak and highly dynamic host-guest complexations and are further reinforced by limited chemical cross-linking for enhanced stability, and then demonstrate the outstanding properties of these Ci-I gelatin hydrogels. The highly dynamic network of Ci-I hydrogels allows injection of prefabricated hydrogels with encapsulated cells and drugs, thereby simplifying administration during surgery. Furthermore, the reversible nature of the weak host-guest cross-links enables infiltration and migration of external cells into Ci-I gelatin hydrogels, thereby promoting the participation of endogenous cells in the healing process. Our findings show that Ci-I hydrogels can mediate sustained delivery of small hydrophobic molecular drugs (e.g., icaritin) to boost differentiation of stem cells while avoiding the adverse effects (e.g., in treatment of bone necrosis) associated with high drug dosage. The injection of Ci-I hydrogels encapsulating mesenchymal stem cells (MSCs) and drug (icaritin) efficiently prevented the decrease in bone mineral density (BMD) and promoted in situ bone regeneration in an animal model of steroid-associated osteonecrosis (SAON) of the hip by creating the microenvironment favoring the osteogenic differentiation of MSCs, including the recruited endogenous cells. We believe that this is the first demonstration on applying injectable hydrogels as effective carriers of therapeutic cargo for treating dysfunctions in deep and enclosed anatomical sites via a minimally invasive procedure.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1. Preparation of the Cell-Infiltratable and Injectable (Ci-I) Gelatin Hydrogels and the Treatment of SAON with Ci-I Hydrogels as the Carrier of Therapeutic Cargoes (Cells and Drug) in the Rat Model

**Figure 1**
(a) Ci-I gelatin hydrogels displayed a frequency-dependent response in the rheological frequency sweep test at a strain of 0.1% and 37 °C. (b) Ci-I hydrogels exhibited a sol–gel transition during switching between alternating high (1000%, unshaded region) and low (1%, shaded region) shear strain in the rheological test at 37 °C. (c) A preformed Ci-I gelatin hydrogel was drawn into a syringe and then injected through a G21 needle into a culture plate well (used as the mold) to be remolded to the shape of the cell culture well. (d) Ci-I gelatin hydrogels sustained over 95% of compressive strain without rupture before or after injection. (e) Stress vs strain curves from a cyclic compression test of Ci-I hydrogels (peak strain, 60%; loading speed, 1 mm/s) at 37 °C (inset: plot in the strain range 50–60%).

**Figure 2**
(a) Kinetics of small-molecule icaritin release from the Ci-I gelatin hydrogels at 37 °C. The confocal micrographs show the 3D distribution of DAPI-stained human mesenchymal stem cell (hMSC) nuclei within the Ci-I hydrogels without (b) or with (c) icaritin encapsulation after 24 h of *in vitro* culture. (d) Schematic illustration of a cell migration experiment at 37 °C. hMSCs seeded on the surface of the icaritin-laden Ci-I gelatin hydrogels infiltrated into the hydrogels within 24 h. (e) Invasion distance of DAPI-stained hMSC nuclei clusters within the Ci-I gelatin hydrogels without icaritin and Ci-I gelatin hydrogels with icaritin. The thickness of the Ci-I hydrogels used for the cell migration test is 1 mm, and the scanning depth of the Ci-I hydrogels is 500 μm in the cell migration experiments. Scale bar: 100 μm (parts b and c).

**Figure 3**
(a) Cell viability staining (green corresponds to live cells, and red corresponds to dead cells) of MSC-laden Ci-I gelatin hydrogels after 1 week and 2 weeks of culture. (b) Gene expression of selected osteogenic markers (alkaline phosphatase (ALP) and Runx2) and adipogenic markers (CEBPα and PPARγ) of MSCs encapsulated in Ci-I gelatin hydrogels after 7 and 14 days of culture. Each sample was internally normalized to GAPDH, and every group was compared to the expression levels of group 100Dex on day 7, the quantitative value of which was set to unity. The data were analyzed with two-way ANOVA with Tukey’s *post hoc* test (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, compared to the 500Dex group). (c) Von Kossa staining; (d) immunohistochemical staining of *Runx2*, *OCN*, *CEBP*α, and *PPAR*γ of the hMSCs loaded in Ci-I gelatin hydrogels after 7 and 14 days of culture. 100Dex, 100 nM dexamethasone (Dex) in media; 500Dex, 500 nM Dex in media; 500Dex+icaritin (M), 500 nM Dex and 1 mM icaritin in media; 500Dex+icaritin (H), 500 nM Dex in media and 1.3 mM icaritin in the hydrogel. The total amount of icaritin in the 500Dex+icaritin (M) group is the same as that in the 500Dex+icaritin (H) group. Scale bar: 100 μm (parts a, c, and d).

**Figure 4**
(a) Micro-CT images and HE staining of the native bone around the tunnel and the new bone inside the tunnel after 3 and 6 weeks postinjection of Ci-I hydrogels in SAON rats. Quantitative analysis of micro-CT for the bone in the “peri-tunnel” (b) and “within tunnel” (c) space. One-way ANOVA with Tukey’s *post hoc* tests were used to analyze the data (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001). (d) Goldener’s Trichrome staining of the bonelike tissue within the bone tunnel. Scale bar: 500 μm (micro-CT images in part a); 100 μm (HE staining in part a and Goldener’s staining in part d).

**Figure 5**
(a) Quantitative analysis of cells positive for Ki67 (Figure S4 for corresponding images) and (b) cells positive for both Osterix and Runx2 using immunofluorescent staining. The data were analyzed with one-way ANOVA with Tukey’s *post hoc* test (n = 4). (c) Immunofluorescent staining at week 6 after SAON surgical treatment in SD rats (negative control group in Figure S5). Representative Western Blot images from three independent experiments for osteogenic markers (osteocalcin and active β-catenin) (d), and adipogenic markers (CEBPα and PPARγ), proliferative marker (PCNA), inflammatory marker (tyrosine-kinase transmembrane receptor for CSF1), and indicator for osteonecrosis (c-Src) (e) (n = 3 per condition; Lane 1, blank repair; Lane 2, Gel; Lane 3, icaritin+Gel; Lane 4, icaritin+MSC+Gel). Injection of Ci-I gelatin hydrogels, which were loaded with both MSCs and icaritin (“icaritin+MSC+Gel”), activated β-catenin and osteocalcin signaling but inhibited the inflammatory response and adipogenesis. The quantitative data are shown in Figure S6. (f) Sequential labeling of newly formed bone with calcein green (CG) and xylenol orange (XO) in the methyl methacrylate-embedded icaritin+MSC+Gel sample at week 3 postsurgery. OB, old bone; T, bone tunnel. (g) Representative images of CG and XO staining (n = 4 images per group) for calculating the bone formation rate. OB, old bone; T, bone tunnel. The quantitative analyses of the bone formation rate at week 3 (h) and week 6 (i) postsurgery were obtained by analyzing the images in part g. The data were analyzed with one-way ANOVA with Tukey’s *post hoc* test (n = 4). ***P < 0.001; scale bar, 100 μm (part c).

**Figure 6**
(a) Prussian blue and nuclear fast red staining of the histological sections of the femoral heads treated with MSCs and icaritin-laden Ci-I hydrogels. SPIO positive cells are indicated by green arrows. SPIO positive cells within the tunnel (b) and around the tunnel (c) were counted. The data were analyzed with one-way ANOVA with Tukey’s *post hoc* test (n = 6, ***P < 0.001).

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References

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[1] Huang Q.; Zou Y.; Arno M. C.; Chen S.; Wang T.; Gao J.; Dove A. P.; Du J. Hydrogel scaffolds for differentiation of adipose-derived stem cells. Chem. Soc. Rev. 2017, 46 (20), 6255–6275. 10.1039/C6CS00052E. - DOI - PubMed

[2] Huang Q.; Zou Y.; Arno M. C.; Chen S.; Wang T.; Gao J.; Dove A. P.; Du J. Hydrogel scaffolds for differentiation of adipose-derived stem cells. Chem. Soc. Rev. 2017, 46 (20), 6255–6275. 10.1039/C6CS00052E. - DOI - PubMed

[3] Lv S.; Wu Y.; Cai K.; He H.; Li Y.; Lan M.; Chen X.; Cheng J.; Yin L. High Drug Loading and Sub-Quantitative Loading Efficiency of Polymeric Micelles Driven by Donor–Receptor Coordination Interactions. J. Am. Chem. Soc. 2018, 140 (4), 1235–1238. 10.1021/jacs.7b12776. - DOI - PubMed

[4] Lv S.; Wu Y.; Cai K.; He H.; Li Y.; Lan M.; Chen X.; Cheng J.; Yin L. High Drug Loading and Sub-Quantitative Loading Efficiency of Polymeric Micelles Driven by Donor–Receptor Coordination Interactions. J. Am. Chem. Soc. 2018, 140 (4), 1235–1238. 10.1021/jacs.7b12776. - DOI - PubMed

[5] Hillel A. T.; Unterman S.; Nahas Z.; Reid B.; Coburn J.; Axelman J.; Chae J. J.; Guo Q.; Trow R.; Thomas A. Photoactivated Composite Biomaterial for Soft Tissue Restoration in Rodents and in Humans. Sci. Transl. Med. 2011, 3 (93), 93ra67.10.1126/scitranslmed.3002331. - DOI - PMC - PubMed

[6] Hillel A. T.; Unterman S.; Nahas Z.; Reid B.; Coburn J.; Axelman J.; Chae J. J.; Guo Q.; Trow R.; Thomas A. Photoactivated Composite Biomaterial for Soft Tissue Restoration in Rodents and in Humans. Sci. Transl. Med. 2011, 3 (93), 93ra67.10.1126/scitranslmed.3002331. - DOI - PMC - PubMed

[7] Rouwkema J.; Rivron N. C.; Van Blitterswijk C. A. Vascularization in tissue engineering. Trends Biotechnol. 2008, 26 (8), 434–441. 10.1016/j.tibtech.2008.04.009. - DOI - PubMed

[8] Rouwkema J.; Rivron N. C.; Van Blitterswijk C. A. Vascularization in tissue engineering. Trends Biotechnol. 2008, 26 (8), 434–441. 10.1016/j.tibtech.2008.04.009. - DOI - PubMed

[9] Speidel A. T.; Stuckey D. J.; Chow L. W.; Jackson L. H.; Noseda M.; Paiva M. A.; Schneider M. D.; Stevens M. M. Multimodal Hydrogel-Based Platform To Deliver and Monitor Cardiac Progenitor/Stem Cell Engraftment. ACS Cent. Sci. 2017, 3 (4), 338–348. 10.1021/acscentsci.7b00039. - DOI - PMC - PubMed

[10] Speidel A. T.; Stuckey D. J.; Chow L. W.; Jackson L. H.; Noseda M.; Paiva M. A.; Schneider M. D.; Stevens M. M. Multimodal Hydrogel-Based Platform To Deliver and Monitor Cardiac Progenitor/Stem Cell Engraftment. ACS Cent. Sci. 2017, 3 (4), 338–348. 10.1021/acscentsci.7b00039. - DOI - PMC - PubMed

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Dynamic and Cell-Infiltratable Hydrogels as Injectable Carrier of Therapeutic Cells and Drugs for Treating Challenging Bone Defects

Affiliations

Dynamic and Cell-Infiltratable Hydrogels as Injectable Carrier of Therapeutic Cells and Drugs for Treating Challenging Bone Defects

Authors

Affiliations

Abstract

Conflict of interest statement

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