Abstract
BACKGROUND:
An enzymatic crosslinking strategy using hydrogen peroxide and horseradish peroxidase is receiving increasing attention for application with in situ-formed hydrogels (IFHGs). IFHGs may also be ideal carrier materials for bone repair, although their ability to carry bone morphogenetic protein-2 (BMP2) has yet to be examined.
OBJECTIVE:
We examined the effectiveness of an IFHG made of hyaluronan (IFHG-HA) containing BMP2 for promoting bone formation in a mouse critical size bone defect model.
METHODS:
C57/BL6J mice received a 2-mm femoral critical-sized bone defect before being randomly assigned to one of the following treatment groups (n = 6): control (no treatment), IFHG-HA only, PBS with BMP2, and IFHG-HA with BMP2. X-ray radiographs were utilized to track new bone formation, and micro-computed tomography and histological examination were performed on new bone formed at the bone defect site two weeks after surgery.
RESULTS:
Mice treated with PBS with BMP2 and IFHG-HA with BMP2 had greater bone volume (BV) and bone mineral content (BMC) than those receiving control, and successfully achieved consolidation. Mice treated with IFHG-HA with BMP2 had significantly higher BV and BMC than those treated with PBS with BMP2.
CONCLUSIONS:
IFHG-HA may be an effective carrier for BMP2 to enable delivery for bone defect repair.
Introduction
There are significant clinical limitations to the rebuilding of large bone segments following extensive bone loss as result of pathological events such as surgical treatment of tumors and trauma. A large proportion of bone defects show signs of healing following administration of current standard bone graft treatments such as autografts, allografts and synthetic grafts. In contrast, these methods may not be effective for healing large defects, or critical bone defects.
Local application of growth factors is used to accelerate bone healing. Bone morphogenetic protein-2 (BMP2), a potent osteoinductive cytokine, can prompt bone and cartilage formation [1–3]. However, because BMP2 is only diffused via local administration, there have been concerns regarding side effects such as attenuation of osteogenic potential and ectopic ossification [4,5]. Therefore, growth factor delivery systems that sustain the release of BMP2 at defect sites are necessary for improving the success of bone healing and to limit possible side effects.
Hydrogels that exhibit rapid gelation, commonly known as in situ-forming hydrogels (IFHGs), are a promising and multifaceted injectable system for tissue engineering applications [5–12]. Previous studies have shown that IFHGs are useful for stem cell-based tissue engineering, cartilage repair, and wound healing. However, it remains unclear whether IFHGs can be used as a carrier for BMP2 to treat bone defects.
Here, we investigated the effectiveness of an IFHG containing a hyaluronan (HA)-tyramine (TA) conjugate for accelerating bone formation in a mouse critical size bone defect model.
Material and methods
In vitro release test
IFHG containing the HA-TA conjugate was purchased from Lifecore Biomedical (Chaska, MN, USA). Recombinant human BMP2 (2 μg) was dissolved in 2% HA-TA solution containing horseradish peroxidase in 0.5 ml Eppendorf tubes. HA-TA/BMP2 solution was subsequently reacted with 4 mM H2O2 solution to obtain a hydrogel. After hydrogel formation, 200 μl of PBS was added to the tube. To determine the BMP2 release profile, BMP2-loaded microtubes were incubated in 200 μl of PBS for 1, 4, 6, 24, 48, 96, 168 (1 week), and 336 (2 weeks) hours.
Animals
All surgeries and handling procedures were conducted in accordance with the Animal Ethics Committee of Kitasato University’s guidelines (Permission number: 2019-127). Twenty-four 10-week-old male C57BL/6J mice (Charles River Laboratories Japan, Inc., Yokohama, Japan) were used for this experiment. The mice were given standard laboratory chow (CRF-1, Oriental Yeast, Tokyo, Japan), and kept in a room with controlled temperature (25 ± 1 °C) and humidity (60 ± 5%) and a 12-hour light/dark cycle. All mice received a 2-mm bone defect created in the center of the right femur, which was fixed with MouseExFix Simple L® (Research Implant System, RIS, Davos, Switzerland), an external device comprising a fixator block and four mounting pins each measuring 0.45 mm in diameter. The mice were subsequently randomly assigned to one of the following treatment groups (n = 6 each): control (no chemical treatment), IFHG (25 μl of IFHG-HA only), BMP2PBS (25 μl of PBS with 2 μg BMP2); and BMP/IFHG-HA (25 μl of IFHG-HA with 2 μg BMP2).
Radiological analysis
At 0, 7, and 14 days after creation of the bone defect, an X-ray system (SOFTEX-CMB4; SOFTEX Corporation, Kanagawa, Japan) was used to track new bone formation in all mice under anesthesia. X-rays were taken using the following settings: exposure, 10 seconds; voltage, 25 kV; current, 10 μA; with X-Ray IX Industrial Film (Fuji Photo Film Co., Ltd., Tokyo, Japan).
Micro-computed tomography analysis
Fourteen days after creation of the bone defect, 3D images were obtained of the femur bone of mice in each group using a micro-computed tomography (CT) device (inspeXio SMX-90CT Plus; Shimadzu Corporation, Tokyo, Japan). The following settings were used: tube voltage, 90 kV; tube current, 100 μA; voxel size 20 × 20 × 20 μm. Bone volume (BV, mm3) and bone mineral content were determined via a 3D imaging program (TRI/3D BON; Ratoc System Engineering Co., Ltd., Tokyo, Japan).
Histology
Following micro-CT, femurs were fixed in 4% paraformaldehyde and demineralized in 0.5 M ethylenediaminetetraacetic acid solution for four weeks. The resulting tissue was paraffin-embedded and cut along the long axis of the femur to obtain 4-μm-thick sections, which were subsequently subjected to hematoxylin and eosin (HE) staining. The area of new bone formed at the defect site was quantified using the freehand tracing tool in ImageJ (National Institutes of Health, Bethesda, MD, USA) (n = 6).
Statistical analysis
All statistical analyses were performed using SPSS (version 25.0; SPSS, Chicago, IL, USA). One-way ANOVA followed by Bonferroni’s post-hoc comparisons test was adopted to analyze differences between groups. P < 0.05 was used as an indicator of statistical significance.
Results
BMP2 release profile in IFHG-HA
The in vitro release profile of BMP2 from IFHG-HA is shown in Fig. 1. BMP2 release from IFHG-HA was observed in the first hour. Thereafter, the sustained release rate was moderate at 4.4 ng/h BMP2 across the 2 weeks.
Sustained release of BMP2 from IFHG-HA in vitro. BMP-2 concentration in PBS at different time points. Results are presented as mean ± standard error (SE) (n = 5).
In mice treated with IFHG/BMP and PBS/BMP, new bone was observed at the bone defect site two weeks after surgery (Fig. 2). In contrast, mice treated with IFHG-HA and control exhibited nonunion at the bone defect site two weeks after surgery.
Representative soft x-ray images obtained 2 weeks after creation of bone defects. Representative radiographs obtained 2 weeks after surgery showing untreated and treated femurs with a critical-sized bone defect. (A) Control, (B) in situ-formed hydrogels made of hyaluronan (IFHG-HA), (C) bone morphogenetic protein-2/phosphate buffered saline (BMP/PBS), and (D) BMP/IFHG-HA treatment groups.
We analyzed micro-CT images to evaluate bone formation at defect sites following two weeks of treatment (Figs 3, 4). Compared to sites that received no treatment (control) or treated with IFHG-HA alone, defect sites injected with IFHG-HA/BMP and PBS/BMP showed significantly greater bone volume and bone mineral content (P < 0.001). Further, mice treated with IFHG-HA/BMP showed significantly greater bone volume and bone mineral content than those receiving PBS/BMP2 (P < 0.001).
Representative micro-computed tomography images of mouse femurs taken 2 weeks after surgery. 3D micro-computed tomography images taken 2 weeks after surgery of untreated and treated femurs with a critical-sized bone defect. (A) Control, (B) in situ-formed hydrogels made of hyaluronan (IFHG-HA), (C) bone morphogenetic protein-2/phosphate buffered saline (BMP/PBS), and (D) BMP/IFHG-HA treatment groups. Scale bars indicate 4 mm.
Analysis of micro-computed tomography images showing new bone formation in mouse femurs 2 weeks after surgery. (A) Bone mineral content and (B) bone volume at bone defect sites. Data indicate mean ± standard error (S.E.), n = 6. a. P < 0.05 compared with the control group. b. P < 0.05 compared with the IFHG-HA group. c. P < 0.05 with the PBS/BMP group.
Histological examination was also performed to evaluate bone formation (Fig. 5). Mice treated with IFHG-HA and control exhibited fibrous tissue in defect sites two weeks after surgery (Fig. 5A, B). In contrast, those treated with PBS/BMP and IFHG-HA/BMP showed large amounts of longitudinal trabecular bone at the defect site (Fig. 5C, D). However, mice treated with IFHG-HA/BMP showed significantly greater new bone area than those receiving PBS/BMP2 (Fig. 5E; P < 0.001).
Representative histological sections obtained from the bone defect site 2 weeks after surgery. Images of hematoxylin and eosin-stained sections of femur bone indicate the formation of new bone at the bone defect site. (A) Control, (B) in situ-formed hydrogels made of hyaluronan (IFHG-HA), (C) bone morphogenetic protein-2/phosphate buffered saline (BMP2/PBS), and (D) BMP2/IFHG-HA treatment groups. (E) Area of new bone at bone defect sites. Data indicate mean ± standard error (S.E.), n = 6. a. P < 0.05 compared with the control group. b. P < 0.05 compared with the IFHG-HA group. c. P < 0.05 with the PBS/BMP group. Scale bars indicate 1000 μm.
BMP2 signaling is initiated in the early part of the first phase of bone healing, which can cause an inflammatory response and periosteal activation, and plays an important role at later phases of osteogenesis [5,13,14]. Therefore, long-term sustained release of BMP2 is important for accelerating bone formation in a bone defect. A previous study reported that IFHG-HA exhibited a sustained release of platelet-derived growth factors for a period of 2 weeks and promoted the proliferation of mesenchymal stem cells in vitro [15]. Similarly, we found that IFHG-HA showed a sustained release of BMP2 across a 2-week period in vitro. In addition, IFHG-HA containing BMP2 stimulated more bone formation than PBS containing BMP2. These findings suggest the potential of an IFHG-HA/BMP2 composite as a therapeutic substance for facilitating bone healing in large bone defects.
There were two limitations in this study. First, we observed only 14 days after the surgery. As the outgrowth bone was still immature, long-term observations to reveal whether this immature bone would be resorbed or become mature cortical or cancellous bone are needed. Second, there is a risk that the results obtained from small animal models may not be clinically relevant due to the smaller size of their long bones and thin and fragile cortices, the absence of haversian-type remodeling in the cortex, and the higher BMP2 doses required to promote bone healing in humans [16,17]. Further investigation using large animal models (e.g. sheep, goats) is paramount for confirming our findings.
Conclusion
In conclusion, combining BMP2 with IFHG-HA facilitated the formation of new bone in a mouse critical-sized bone defect model. IFHG-HA/BMP2 composite may be a promising substance for repairing large bone defects.
Footnotes
Conflict of interest
None to report.
Funding
This investigation was supported in part by Grant-in-Aid for Young Scientists Grant No. 19K18546, Grant-in-Aid for Scientific Research (C) No. 18K09079, Grant-in-Aid for Young Scientists (Start-up) No. 20K23008, and a Medical Research Grant from The General Insurance Association of Japan.