The optimal dosage of hyaluronic acid for bone regeneration in rat calvarial defects

- ¹Department of Periodontology and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea.
- ²One-Stop Specialty Center, Seoul National University Dental Hospital, Seoul, Korea.
Correspondence: Ki-Tae Koo. Department of Periodontology and Dental Research Institute, School of Dentistry, Seoul National University, 101 Daehakno, Jongno-gu, Seoul 03080, Korea. Email: periokoo@snu.ac.kr ; Tel: +82-2-2072-0108; Fax: +82-2-744-0051

Received June 20, 2022; Revised September 19, 2022; Accepted October 19, 2022.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/).

Abstract

Purpose

Hyaluronic acid (HA) affects angiogenesis and promotes the migration and differentiation of mesenchymal cells, thereby activating the osteogenic ability of osteoblasts. Although studies on the action of HA during bone regeneration are being actively conducted, the optimal dose of HA required for bone regeneration remains unclear. Therefore, the purpose of this study was to elucidate the most effective HA dose for bone formation using a rat critical-size defect model.

Methods

Thirty rats were randomly divided into 5 groups, with 6 rats in each group. An absorbable collagen sponge soaked with HA or saline was used to fill an 8-mm defect, which was then covered with a collagen membrane. Different treatments were performed for each group as follows: (1) saline control, (2) 1 mg/mL HA, (3) 25 mg/mL HA, (4) 50 mg/mL HA, or (5) 75 mg/mL HA. After a healing period of 4 weeks, micro-computed tomography and histological analysis were performed. The obtained values were analyzed using analysis of variance and the Tukey test (P<0.05).

Results

At week 4, the 75 mg/mL HA group had the highest bone volume/total volume ratio, new bone, and bone fill among the 5 groups, and these values were significantly different from those observed in the control group (P<0.01) and 1 mg/mL HA group (P<0.001). More active bone formation was observed in the higher-dose HA groups (25 mg/mL, 50 mg/mL, and 75 mg/mL HA), which included a large amount of woven bone.

Conclusions

The 75 mg/mL HA group showed better bone formation than the other groups (1, 25, and 50 mg/mL HA and control).

Graphical Abstract

Keywords

Animal models; Bone; Bone regeneration; Hyaluronic acid; Rats

INTRODUCTION

Bone regeneration has become a fundamental concept of dental treatment and is widely used for the restoration of periodontal defects, dental implant therapy, and dental prosthetic treatments [1]. Currently, guided bone regeneration (GBR) therapy is carried out using bone graft materials, barrier membranes, and biologics, and promising bone regeneration results have been obtained [2]. Due to the different materials used in GBR, the bone regeneration capacity varies substantially. Therefore, research is needed to find better substances for promoting bone formation and maturation.

Hyaluronic acid (HA), also called hyaluronan or hyaluronate, is present in the majority of mammalian tissues, including the soft connective tissue, synovial fluid of the joints, the lung, kidney, brain, and muscle tissue [3, 4]. As a glycosaminoglycan, HA is a crucial component of the extracellular matrix that is necessary for a variety of biological functions and plays an essential role in wound healing [5, 6]. During the early bone regeneration process, fibroblasts secrete HA [7]. Previous studies have shown that HA is involved in cellular processes, such as morphogenesis, wound healing, inflammation, and metastasis [5, 8, 9, 10, 11]. Additionally, HA has been shown to affect angiogenesis and can enhance the osteogenic capacity of osteoblasts by promoting the migration and differentiation of mesenchymal cells [12, 13, 14]. HA has low immunogenicity and high biocompatibility, making it especially useful compared to other growth factors or scaffolds used during bone healing [15, 16]. These advantages have drawn attention to the potential of HA as an excellent bone regeneration material.

In recent years, research on the impact of HA on the bone healing process has demonstrated its promise. Studies conducted in both normal and infected sockets showed that HA promotes bone formation and wound healing [17, 18]. A study by Huang et al. [19] showed that HA had dose-dependent and molecular weight-specific effects on cell proliferation, differentiation, and bone formation, particularly at high molecular weights, where alkaline phosphatase activity significantly increased [19]. However, there is an absence of research on the ideal HA dose required for bone healing in vivo. Determining the HA dose required for bone formation and maturation would be enormously helpful for the GBR process. Hence, this study explored the optimal dose of HA for bone regeneration using a rat critical-size defect model.

MATERIALS AND METHODS

Animals

All research protocols were approved by the Institutional Animal Care and Use Committee, Seoul National University, Korea (SNU-190722-2-2). Thirty 11- to 13-week-old male Sprague-Dawley rats (OrientBio, Seoul, Korea) weighing 300–400 g were used for this experiment. The rats were acclimatized for at least 1 week before the experiments. The animals were randomized into 5 groups, each containing 6 animals. Each plastic cage housed 2 animals with tail-marking in the monitoring environment (temperature 23°C; humidity controlled at 60%; 12:12-hour light-dark cycle). They had ad libitum access to water and a standard pellet rat-food diet. All experiments were performed according to the guidelines of the Institute of Laboratory Animal Resources, Seoul National University.

Formulations of HA

The different doses of HA used in this study were obtained by mixing HA with a molecular weight of 1000 kDa (Shiseido Sodium Hyaluronate SZE [Grade-EP], Shiseido, Shizuoka, Japan) and phosphate-buffered saline. The concentrations were as follows: 1 mg/mL, 25 mg/mL, 50 mg/mL, and 75 mg/mL. After mixing, the HA was sterilized at 121°C for 15 minutes.

Surgical procedures

The animals were anesthetized by an intraperitoneal injection of a combination of 25 mg/kg tiletamine/zolazepam (Zoletil 50, Virbac, Carros, France) and 10 mg/kg xylazine hydrochloride (Rompun, Bayer Korea, Ansan, Korea). After anesthesia, the dorsal surface of the head was shaved and disinfected with povidone-iodine. Routine invasive anesthesia was performed at the surgical site with 2% lidocaine HCl and epinephrine (1:100,000, Huons, Seongnam, Korea). A 3-cm midline incision was made through the skin along the sagittal suture of the skull, and the soft tissues and periostea were elevated and reflected (Figure 1A). Under saline irrigation, craniotomy defects with an 8-mm diameter were created in rats using a trephine bur (Figure 1B). An absorbable collagen sponge (ACS) (Collagen Graft 2, Genoss, Suwon, Korea) cut into the shape of the defect with a diameter of 8 mm was prepared in advance (Figure 1C). The rats were divided into 5 groups as follows: (1) control group, the defect area (DA) received an ACS soaked with saline; (2) 1 mg/mL HA group, the DA received an ACS soaked with 1 mg/mL HA; (3) 25 mg/mL HA group, the DA received an ACS soaked with 25 mg/mL HA; (4) 50 mg/mL HA group, the DA received an ACS soaked with 50 mg/mL HA; (5) 75 mg/mL HA group, the DA received an ACS soaked with 75 mg/mL HA. The collagen soaked in HA or saline was placed in the defect site (Figure 1C and D), and a collagen membrane (Collagen Membrane-P, Genoss) was used to cover the surgical site (Figure 1E). The incision was sutured in layers with 4-0 Vicryl (Ethicon, Raritan, NJ, USA) and 5-0 Monosyn (B. Braun Surgical, SA Rubi, Spain) (Figure 1F). All rats received intramuscular injections of antibiotics (cefazolin, Chong Kun Dang, Seoul, Korea) and analgesics (Meloxicam, Labina, Barcelona, Spain) directly after surgery. Four weeks after surgery, the rats were sacrificed with excess CO₂ gas.

Figure 1
Surgical procedures. (A) A 3-cm midline incision was made through the skin along the sagittal suture of the skull, and the soft tissues and periostea were elevated and reflected. (B) Under saline irrigation, craniotomy defects with an 8-mm diameter were created in rats using a trephine bur. (C) An absorbable collagen sponge was cut into the shape of the defect with a diameter of 8 mm, soaked in hyaluronic acid or saline, and (D) placed in the defect site. (E) A collagen membrane was used to cover the surgical site. (F) The incision was sutured in layers.

Micro-computed tomography (micro-CT)

Tissues, including the surgical sites, were harvested and fixed in 10% neutralized buffered formalin, and micro-CT images were taken using a micro-CT device (SkyScan 1173, Bruker, Kontich, Belgium) at a pixel size of 9.94 μm (130 kV, 60 μA). Micro-CT data were measured 3 times by a single blinded and calibrated examiner (LL) using the same software (CTan, Bruker). Three-dimensional visualization images were obtained using CTVox software (Bruker). The bone volume/total volume ratio (BV/TV) was measured for each defect with a gray threshold level of 52–250. In the defect field, a cylindrical area with a thickness of 0.7 mm from the base along the original defect margin and a diameter of 8 mm was defined as the volume of interest.

Histological preparation and analysis

The specimens were decalcified with a 10% ethylenediaminetetraacetic acid solution for 2 weeks before they were dehydrated by a series of ethanol solutions of increasing concentrations and embedded in paraffin. A coronal section with a thickness of 5 μm through the center of the circular defect was obtained and stained with hematoxylin and eosin. The prepared specimens were examined by light microscopy. After a microscopic examination, a photograph of each slide was taken using a digital slide scanner (PANNORAMIC 250 Flash III, 3DHISTECH, Budapest, Hungary), and the resulting images were saved on a computer for analysis. A single blinded, calibrated examiner examined all of the images 3 times using the same software (ImageJ 1.53e, National Institutes of Health, Bethesda, MD, USA). The following parameters were recorded for each defect site:

• DA: the total DA was defined as the area surrounded by 2 defect margins and 2 phantom lines drawn along the inner and outer calvarial bone contours
• New bone area (NB): the total area of newly formed bone in the DA
• Bone fill: the fraction (%) of newly formed bone within the DA

Statistical analysis

All data are expressed as the mean ± standard deviation. Statistical analyses were performed using statistical software (SPSS version 25, IBM, Armonk, NY, USA). One-way analysis of variance followed by the Tukey post hoc test was used to determine the significance of mean differences between groups. A P value less than 0.05 was considered to indicate statistical significance. The sample size was calculated based on a previous study considering 2% of new bone formation as clinically relevant, with a standard deviation of 1% and allocating 6 animals in each group (α=0.05, and β=0.2) [20].

RESULTS

All 30 defect sites healed well with no significant signs of infection, inflammation, or postoperative bleeding. No noteworthy events occurred during the experiments.

Micro-CT analysis and morphometric evaluation

Figure 2 shows the BV/TV results obtained from micro-CT of the calvaria. The BV/TV values were 19.73±11.25 in the control group, 14.63±9.81 in the 1 mg/mL HA group, 30.22±8.27 in the 25 mg/mL HA group, 25.69±10.96 in the 50 mg/mL HA group and 40.03±13.88 in the 75 mg/mL HA group. The 75 mg/mL HA group had a significantly higher BV/TV than the control group and the 1 mg/mL HA group (P<0.001) (Figure 3). The BV/TV values also generally tended to increase with increasing HA doses (Figure 3). In the micro-CT analyses, it could be observed that the defects in all the groups were partially closed (Figure 2). The greatest amount of new bone formation was observed in the 75 mg/mL HA group (Figure 2E, J and O). New bone formation around the defect margin was denser and thicker than that in the middle part of the defect.

Figure 2
Micro-computed tomography analysis of newly formed bone at the calvarial defects in the control group, 1 mg/mL HA group, 25 mg/mL HA group, 50 mg/mL HA group, and 75 mg/mL HA group at 4 weeks postoperatively. Sectional images with 3-dimensional reconstruction images. (A-E) Transverse view, (F-J) coronal view, and (K-O) 3-dimensional reconstruction. The orange area in the image indicates the volume of interest. Notably, the 75 mg/mL HA group exhibited the most new bone formation among the 5 groups. The control and 1 mg/mL HA groups showed less new bone formation.
HA: hyaluronic acid.

Figure 3
Micro-computed tomography quantitative analysis of newly formed bone at the calvarial defects. The BV/TV ratio at 4 weeks after defect creation is shown. Notably, the 75 mg/mL HA group induced significantly more bone formation compared with the control and 1 mg/mL HA groups.
HA: hyaluronic acid, BV/TV: bone volume/total volume.

^a)P<0.01.

Histological observations

Figure 4 shows histological images 4 weeks after defect creation. There was no sign of inflammation or infection in any group. The defect sites in each group were partially filled with mineralized bone, and the collagen membrane and ACS residues were not completely absorbed. More active bone formation was observed in the relatively high-dose HA groups (25 mg/mL, 50 mg/mL, 75 mg/mL HA groups), and it could be seen that they contained a large amount of woven bone. Additionally, this newly formed bone was more apparent at the margins than at the center of the defects. The 75 mg/mL HA group showed more blood vessels than any other group.

Figure 4
Hematoxylin and eosin-stained histological sections at 4 weeks after defect creation. Representative histological sections show a cross-section of the entire defect with native bone at the edges. (A) Some newly formed bone was observed at the defect site in the control group, which was relatively small. (B) New bone was observed at the surgical margins of the 1 mg/mL HA group, which had the least amount of bone formation among the 5 groups. (C, D) Somewhat more new bone was formed at the defect in the 25 mg/mL and 50 mg/mL groups. (E) Of all groups, the 75 mg/mL HA group showed the most new bone and blood vessels compared to the other groups.
HA: hyaluronic acid.

Histomorphometric analysis

The results of the histomorphometric analysis are shown in Table 1 and Figure 5. There were no statistically significant differences in DAs among the 5 groups. The 1 mg/mL HA group had less NB and less bone fill than the control group, but without a statistically significant difference. The 25 mg/mL HA group and the 50 mg/mL HA group had similar NB and bone fill, which were both greater than those of the control group. However, the differences were not statistically significant. The 75 mg/mL HA group had the greatest NB and bone fill among the 5 groups, with values that were significantly higher than those of the control (P<0.01) and 1 mg/mL HA groups (P<0.01).

Table 1
Histomorphometric results for the 5 study groups at 4 weeks postoperatively

Click for larger image
Click for full table
Download as Excel file

Figure 5
Histomorphometric analysis of new bone formation in the defects. (A) NB and (B) bone fill at 4 weeks after defect creation are shown. The NB and bone fill were significantly higher in the 75 mg/mL HA group than in the control and 1 mg/mL HA groups.
HA: hyaluronic acid, NB: new bone area.

^a)P<0.01.

DISCUSSION

This study was conducted to find the optimal dose of HA for bone regeneration. A critical defect size of 8 mm [20] was used to analyze the appropriate HA dose, and a collagen membrane was used to increase the stability of the defect site. Four weeks after the critical defect was created in rat calvaria, the dose of 75 mg/mL showed the best bone healing compared to the other groups.

There is a close relationship between the dose of HA and cell proliferation and differentiation [21]. Previous in vitro studies have shown increased cell growth at higher HA doses [19, 22, 23]. However, Kaneko et al. [24] observed that a high dose of HA strongly inhibited the development of mouse myoblastic cells and bone marrow cells into osteoblasts. These results suggest that finding an appropriate threshold for the osteoinductive dose is very important in the process of bone healing. In the present study, the degree of bone regeneration varied with the dosage (Figure 2). Among the 5 groups used in the study (1, 25, 50, and 75 mg/mL HA, and control), the highest BV/TV was observed at 75 mg/mL. In addition, it was generally shown that with increasing doses of HA, newly formed bone also increased. A similar result was obtained from histological observations. In the histological evaluation, as the dosage of HA increased, more bone formation dispersed in a large area of the defect site was observed, especially in the 75 mg/mL HA group.

Histological analyses showed a large area of woven bone with more blood vessels at higher HA doses (especially 75 mg/mL). This may have been influenced by the angiogenesis-promoting function of HA [25]. HA is indirectly involved in bone wound healing by stimulating angiogenesis [7, 26]. Previous studies have also demonstrated the effect of HA on the differentiation of mesenchymal cells into osteoblasts [19]. Through these mechanisms, HA is believed to have a beneficial effect on improving the wound healing process.

Previous experiments conducted by Bezerra et al. [1] showed that in 5-mm rat calvarial defects, the use of 1% HA gel and ACS resulted in more bone fill than ACS alone and blood clots. However, when only 1% HA was used, bone formation did not improve [1]. In our study, there was no significant difference in bone formation in the control group compared with the 1, 25, or 50 mg/mL HA + ACS groups; the only significant difference was found for the 75 mg/mL HA group. This finding indicates that ACS is a suitable scaffold for bone formation. Additionally, if used with an appropriate dose of HA, a synergistic effect can be obtained during bone regeneration.

Other studies have used HA in combination with other biomaterials. Huang et al. [27] reported that HA promotes osteogenic and angiogenetic activity when used in combination with bone morphogenetic protein 2 (BMP-2)/ACS. In another study, a thiol-modified hyaluronan hydrogel showed better osteogenic capacity than ACS when used in combination with BMP-2 [28]. In the above 2 experiments, the combined use of HA increased the osteogenic potential and reduced the side effects of BMP-2. This is due to the fact that HA enables the gradual, continuous release of BMP-2, which can promote bone growth over a longer time [27, 28, 29]. In addition, several previous studies have demonstrated that using HA in conjunction with bone grafts can improve bone regeneration [30, 31, 32].

In vivo experiments using different doses of HA have been rarely conducted because most studies used commercially available HA products. Therefore, those articles have not specifically mentioned the HA concentration or dosage. Therefore, the significance of this study is that different concentrations of HA were specifically prepared to perform the experiments. To the authors’ knowledge, few studies have yet attempted this, although this information would greatly contribute to the study of the relevance of HA to bone regeneration. However, the present work has several flaws, chief among them the inability to quantify the amount of HA and saline soaked into the ACS. HA may be lost due to suction or spread to other sites during surgery, which may have had a certain impact on the experimental results. Additionally, the effect of a higher dose of HA than 75 mg/mL on bone healing requires research. Therefore, we plan to add higher-dose HA groups in a follow-up study to determine the osteoinductive dose threshold. Within the limits of this study, it can be concluded that the 75 mg/mL HA group showed better bone formation than the other groups (1, 25, and 50 mg/mL HA and control). Additional research is required to investigate osteoinductive dose thresholds using higher doses of HA and to observe bone maturation with longer healing periods.

Notes

Funding:The project was supported by a grant from the GENOSS (NCR19008), Republic of Korea.

Conflict of Interest:No potential conflict of interest relevant to this article was reported.

Author Contributions:

Conceptualization: Ki-Tae Koo, Ling Li.
Formal analysis: Ling Li, Jungwon Lee.
Investigation: Ling Li, Young-Dan Cho.
Methodology: Jungwon Lee, Yong-Moo Lee, Young-Dan Cho.
Project administration: Ki-Tae Koo, Sungtae Kim, Yang-Jo Seol.
Writing - original draft: Ling Li.
Writing - review & editing: Ki-Tae Koo, Jungwon Lee, Sungtae Kim, Yang-Jo Seol, Yong-Moo Lee.

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