In situ-formed hyaluronan gel/BMP-2/hydroxyapatite composite promotes bone union in refractory fracture model mice

2.1. Synthesis of graft materials

We produced our hyaluronan-tyramine (HyA-TA) conjugate according to methods described previously [14]. To synthesize IFGs-HyA, we crosslinked the HyA-TA polymer in the presence of HRP (FUJIFILM Wako Pure Chemical Corporation; pH 7.4) and H 2 O 2 in 10 mM phosphate-buffered saline (PBS; pH 7.4). Subsequently, Hap (10 mg) was dissolved in 2% HyA-TA polymer solution together with 0.8 units/mL HRP and 2 μg of BMP-2 (PEPROTECH, Inc. Rocky Hill, NJ, USA). The curing reaction was initiated by adding 4 mM H 2 O 2 to a solution of BMP-2/HyA-TA/HRP/Hap.

2.2. Refractory fracture model mice

This study was conducted on 9-week-old C57BL/6J mice [15]. The mice were kept at Nippon Charles River Laboratories (Kanagawa, Japan) in a semi-barrier system under optimal and controlled temperature, humidity and light conditions, and were provided standard rodent chow (CRF-1; Oriental Yeast, Tokyo, Japan).

All surgical procedures were performed under sterile conditions. To generate the fracture model, first, a scalpel was used to make a 10-mm incision into the lateral side of the left thigh. Second, a 4-mm lateral parapatellar incision was made to dislocate the patella medially. Third, a drill was used to make a 0.5-mm hole in the intracondylar notch to enable a stainless steel needle (0.5 mm in diameter) to subsequently be retrogradely inserted into the canal within the intramedullary cavity. Fourth, the osteotomy procedure was performed with a wire saw (0.22 mm in diameter) via a small lateral approach. A stainless steel needle was then inserted into the intramedullary canal through a small incision to stabilize the fracture. Fifth, to create an intractable fracture, a bipolar electrocautery was applied at 1.2 W of power for 10 seconds to erode the whole perimeter of a 3-mm-wide section of periosteum with the fracture site at the center, excluding the medullary region. Immediately following the fracture, 25 μl of IFGs-HyA/Hap harboring 2 μg BMP-2 (IFGs-HyA/Hap/BMP-2) or Hap harboring 2 μg BMP-2 (Hap/BMP-2) was introduced into the region around the fracture (Fig. 1A, B). We ensured that the whole injury site was covered and that the area surrounding the grafted site was subsequently stabilized for each treatment group (n = 10 each). Animals that underwent the fracture surgery but did not receive any treatment were considered the control group (n = 10). Micro-CT and histological analyses were conducted on the femurs of a total of 10 mice in each of the following groups: IFGs-HyA/Hap/BMP-2, Hap/BMP-2 and control. All analyses were conducted in a blinded fashion, such that the investigators remained unaware of the group assignment of each mouse. All animal experiments were conducted based on the guidelines of the Animal Ethics Committee of Kitasato University (Approval numbers: 2020-090, 2021-045, and 2022-121).

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Fig. 1.

Graft of bone morphogenetic protein-2 (BMP-2)/in situ-formed hyaluronic acid hydrogels (HyA)/hydroxyapatite (Hap) composite in the fracture site. (A) Fracture was created in the mice femur. (B) After creation of the fracture, BMP-2/Hya/Hap composite was immediately introduced into the region around the fracture. White arrows indicate the fracture site. Scale bar = 10 mm.

A micro-focus X-ray CT system (inspeXio SMX-90CT; Shimadzu, Tokyo, Japan) was used to measure the bone volume (BV) and bone mineral content (BMC) of a defined region of interest. BV and BMC were used as indicators of new bone formation [15,16].

2.4. Histology

After micro-CT analysis, femur bones were submerged in decalcifying solution (KCX, Falma, Tokyo, Japan) and then subjected to paraffin embedding. The blocks of tissue were sectioned at a thickness of 4 μm using a microtome (RETORATOME REM-710; YAMATO KOHKI, Saitama, Japan) and the resulting tissue sections were stained with hematoxylin and eosin. We subsequently used the freehand tracing tool in ImageJ version 1.52 (National Institutes of Health, Bethesda, MD) to outline and quantify the area of new bone generated within a 3.5-mm region of interest correlating to the cauterized region with the fracture site at the center.

3.1. IFGs-HyA/Hap gel harboring BMP-2 induced callus formation

Using micro-CT imaging analysis, we evaluated the formation of calluses on fractured femurs 4 weeks after surgery (Fig. 2A-C). Comparison of fracture sites that were untreated (control) with those treated with IFGs-HyA/Hap/BMP-2 or Hap/BMP-2 showed that IFGs-Hya/Hap/BMP-2 and Hap/BMP-2 significantly elevated BV and BMC (p < 0.001, Fig. 3A, B). Comparison of IFGs-Hya/Hap/BMP-2 with Hap/BMP-2 showed that IFGs-Hya/Hap/BMP-2 had a significantly greater impact on increasing BV and BMC (p < 0.001, Fig. 3A, B).

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Fig. 2.

Representative 3D micro-computed tomography images of femurs 4 weeks after surgery. (A) Control, (B) Hap/BMP-2, and (C) IFGs-HyA/Hap/BMP-2-treated groups. IFGs-HyA: in situ-formed gels consisting of hyaluronan; Hap: hydroxyapatite; BMP-2: bone morphogenetic protein-2.

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Fig. 3.

Bone volume and bone mineral content at sites of fracture 4 weeks after surgery. Analysis was conducted on micro-computed tomography images of femurs to examine new bone formation 4 weeks after surgery in the control (white bars), Hap/BMP-2 (gray bars), and IFGs-HyA/Hap/BMP2 (speckled bars) groups. (A) Bone volume, (B) bone mineral content. n = 10. ^∗ P < 0.05.

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Fig. 4.

Representative histological images of the fracture site 4 weeks after surgery. Sections through the femur were stained with hematoxylin and eosin to reveal new bone formation at the fracture site. (A) Control, (B) HA/BMP-2, and (C) IFGs-HyA/Hap/BMP2-treated groups. Areas enclosed by yellow dotted lines indicate newly formed bone. Scale bars indicate 500 μm. (D) Quantification of the area of new bone formed at fracture sites. Control (white bar), HA/BMP-2 (gray bar), and IFGs-HyA/Hap/BMP-2 (speckled bar) groups are shown. n = 10. ^∗ P < 0.05.

A histological examination was also conducted four weeks after the fracture to assess bone union. Compared with the control group, mice treated with IFGs-HyA/Hap/BMP-2 showed larger calluses at the fracture site. The newly formed bone created a bridge around the perimeter of the fracture site (Fig. 4). In contrast, in the Hap/BMP-2 and control groups, we noted fibrous or cartilage callus tissue at the fracture site, with no indications of bone union (Table 1).