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. 2021 Nov 25:9:723789.
doi: 10.3389/fcell.2021.723789. eCollection 2021.

Recombinant Human Bone Morphogenic Protein-2 Immobilized Fabrication of Magnesium Functionalized Injectable Hydrogels for Controlled-Delivery and Osteogenic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells in Femoral Head Necrosis Repair

Xueliang Lu  1 , Hongyu Guo  2 , Jiaju Li  2 , Tianyu Sun  2 , Mingyue Xiong  1
Affiliations

Recombinant Human Bone Morphogenic Protein-2 Immobilized Fabrication of Magnesium Functionalized Injectable Hydrogels for Controlled-Delivery and Osteogenic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells in Femoral Head Necrosis Repair

Xueliang Lu et al. Front Cell Dev Biol. .

Abstract

Femoral head necrosis (FHN) is a clinically progressive disease that leads to overwhelming complications without an effective therapeutic approach. In recent decades, transplantation of mesenchymal stem cells (MSCs) has played a promising role in the treatment of FHN in the initial stage; however, the success rate is still low because of unsuitable cell carriers and abridged osteogenic differentiation of the transplanted MSCs. Biopolymeric-derived hydrogels have been extensively applied as effective cell carriers and drug vesicles; they provide the most promising contributions in the fields of tissue engineering and regenerative medicine. However, the clinical potential of hydrogels may be limited because of inappropriate gelation, swelling, mechanical characteristics, toxicity in the cross-linking process, and self-healing ability. Naturally, gelated commercial hydrogels are not suitable for cell injection and infiltration because of their static network structure. In this study, we designed a novel thermogelling injectable hydrogel using natural silk fibroin-blended chitosan (CS) incorporated with magnesium (Mg) substitutes to improve physical cross-linking, stability, and cell osteogenic compatibility. The presented observations demonstrate that the developed injectable hydrogels can facilitate the controlled delivery of immobilized recombinant human bone morphogenic protein-2 (rhBMP-2) and rat bone marrow-derived MSCs (rBMSCs) with greater cell encapsulation efficiency, compatibility, and osteogenic differentiation. In addition, outcomes of in vivo animal studies established promising osteoinductive, bone mineral density, and bone formation rate after implantation of the injectable hydrogel scaffolds. Therefore, the developed hydrogels have great potential for clinical applications of FHN therapy.

Keywords: cell encapsulation; femoral head necrosis; injectable hydrogel; magnesium; stem cells.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic representation of magnesium (Mg) loaded chitosan (CS)/silk fibroin (SF) blended injectable hydrogel formation with probable mechanisms according to the present investigation.
FIGURE 2
FIGURE 2
Structural interactions, chemical compositions, and elemental distributions were evaluated by spectroscopic methods of Fourier transform infrared (FTIR) (A), x-ray diffraction (XRD) (B), and XPS (C) analyses.
FIGURE 3
FIGURE 3
Morphological structure, porous network, and component distributions were investigated and presented using scanning electron microscope (SEM) and transmission electron microscope (TEM) microscopic instruments and EDX mapping results; (A1–3) are the SEM observations of the CS, magnesium loaded chitosan/silk fibroin (Mg/CSSF) and, Mg/CSSF hydrogel groups, respectively, which exhibit improved porous morphology consistent with the pore size analysis results (A4). Transmission microscopic observations of hydrogel groups are displayed as (B1) CS, (B2) Mg/CSSF, (B3) Mg/CSSF, and (B4) SAED patterns of Mg/CSSF. (C) Elemental distributions of C, O, N, and Mg elements were confirmed by EDX mapping analysis.
FIGURE 4
FIGURE 4
Swelling ratio (A), biodegradation behaviors (B), and in vitro recombinant human bone morphogenic protein-2 (rhBMP-2) release profiles (C,D) of fabricated injectable hydrogel groups [CS, chitosan/silk fibroin (CSSF), and Mg/CSSF] for different time durations, as required for experimental accuracy.
FIGURE 5
FIGURE 5
Systematic observations of temperature dependent G′ and G′′ functions for the (A1) CS, (A2) Mg/CSSF, and (A3) Mg/CSSF hydrogel groups. (B1,2) Viscosity vs. shear rate observations of prepared hydrogel groups at 25 and 37°C, respectively. (C) Stress–strain curve results of the hydrogel groups.
FIGURE 6
FIGURE 6
(A) Live/Dead cell morphology and proliferations of the rat bone marrow-derived MSCs (rBMSCs) cultured with prepared hydrogel groups observed under confocal laser scanning microscopy (CLSM) at different incubation times (1, 3, and 7 days). (B) Cell seeding efficiency, (C) cell survival absorbance, (D) proliferation rate, and (E) live cell number of rBMSCs encapsulated in hydrogel groups for different incubation durations. ****p < 0.0001; ***p = 0.0001–0.001; **p = 0.001–0.01; *p = 0.01–0.05; p ≥ 0.05 means not significant (NS).
FIGURE 7
FIGURE 7
Analysis of osteogenic differentiation efficacy of rBMSCs on injectable hydrogel groups. (A,B) Alkaline phosphatase (ALP) activity at different incubation times of 7 and 14 days, respectively. (C) Quantitative analysis of osteogenic gene expressions of rBMSCs seeded on hydrogel groups at day 14 of the culture period (n = 3, *p < 0.05, ***p value 0.0001–0.001 of significant differences).
FIGURE 8
FIGURE 8
(A) Radiography (x-ray) observation of femoral defects in RD rat models transplanted with rBMSCs encapsulated rBMSC@rhBM-Mg/CSSF and other comparative hydrogel groups (no implant defect models considered as control). Quantitative investigations of (B) bone volume (mm3), (C) radiographic score, and (D) bone mineral content (BMC) on hydrogel treated in vivo models. **p = 0.001–0.01.
FIGURE 9
FIGURE 9
Histological sections of treated femoral defect models implanted with injectable hydrogel groups were observed by using hematoxylin and eosin (H&E) (A) and Masson’s trichrome (MTS) stains (B). The histological microscopic observations were observed in two magnifications (10× and 40×) to show bone remodeling and regeneration.
FIGURE 10
FIGURE 10
In vivo biosafety assessments: (A) body weight changes of RD rats after implantation of hydrogel groups according to postimplantation time (weeks). (B) Quantitative observations of average value of Mg2+ ions concentrations in the different organs of treated models after 8 weeks and (C) histological observations of treated models’ microstructures of heart, liver, lung, kidneys, and spleen with H&E staining.
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