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J Vet Clin 2022; 39(6): 302-310

https://doi.org/10.17555/jvc.2022.39.6.302

Published online December 31, 2022

Effect of Keratin-Based Biocomposite Hydrogels as a RhBMP-2 Carrier in Calvarial Bone Defects Mouse Model

Jongjin Lee , Jinsu Kang , Jaewon Seol , Namsoo Kim , Suyoung Heo

College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, Korea

Correspondence to:*syheo@jbnu.ac.kr

Received: August 12, 2022; Revised: October 14, 2022; Accepted: November 23, 2022

Copyright © The Korean Society of Veterinary Clinics.

Recently, in human medicine and veterinary medicine, interest in synthetic bone graft is increasing. Among them, bone morphogenic protein (BMP) is currently being actively researched and applied to clinical trials. However, BMP has the disadvantage of being expensive and easily absorbed into surrounding tissues. Therefore, BMP requires the use of small amounts and rhBMP (recombinant human bone morphogenetic protein)-2 carriers that can be released slowly. Hydrogel has the property of swelling a large amount of water inside when it is aqueous solution, and when it is, it consists of more than 90 percent water. Using these properties, hydrogels are often used as rhBMP-2 carrier. The scaffold used in this study is a hydrogel made from which keratin is extracted using human hair and based on it. In this study, we wanted to see the effect of bone formation in the calvarial defect model by using keratin-based hydrogel made with human hair as a scaffold. The experiment was conducted by dividing 3 groups a total of 12 mice. Calvarial bone defect is set to all 4 mm diameters. Bone formation was evaluated by using gross evaluation, micro-computed tomography (micro-CT), immunohistochemistry. Groups using keratin-based hydrogel were significantly observed compared to Group 1s, and the most bone formations were found when rhBMP-2 and hydrogel were used. This represents the superiority of the functions of the rhBMP-2 carrier by a new material, keratin-based hydrogel. Through gross evaluation, micro-CT, and immunohistochemistry, we can confirm that keratin-based hydrogel is a useful rhBMP-2 carrier.

Keywords: keratin, hydrogel, rhBMP-2 carrier, calvarial defect model, mouse.

In the field of orthopedics and neurosurgery, bone transplants are required in many cases. Bone transplants are required in the reconstruction of congenital deficits and acquired lesions, or deficits caused by trauma. In addition, bone transplants are often essential in the number of dental fields in patients with severe bone absorption following the onset of chronic periodontitis. Forming a new bone is the domain of tissue engineering. Three major points of tissue engineering are the source of cells, matrix or scaffold, and signal moles. In terms of bone regeneration, this can be seen as osteoplastic cells, hormones with bone-inducing effects, growth factors, and substrate with bone conduction effects.

A lot of progress in the field of veterinary medicine is autograft, which is considered gold standard. However, it tends to be easy absorbed, and if a small dog’s bone graft is carried out, a fracture, a small amount of bone sample is sometimes a problem. Xenograft bone transplants that use tissues from different species have problems with immune response. Therefore, the field where much research has been conducted in human medicine and veterinary medicine recently is according to synthetic bone transplantation. This has the advantage of not having to perform any additional surgery on the other side of the body and being able to transplant to the extent planned by the surgeon. There is platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), bone morphogenetic proteins (BMP)-2, BMP-7 and insulin-like growth factors (IGF) I, II of this kind (1,20).

Among them, BMP is currently being actively researched and applied to clinical trials. BMP is involved in osteogenesis and chondrogenesis and is also involved in embryonic development and fracture healing. The mechanism at the cell level works in binding with the receptor on the surface of the cell. When BMP is coupled to a receptor, cascade through the serine/threonine kinase is expressed in the cell. After this process, the differential hepatocytes are differentiated into osteoporosis. It also allows the production of vascular endothelial group factor-α (VEFG-α) from osteoporosis to help produce angiogenesis essential to bone formation (10).

Osteocytes consume more oxygen than mesenchymal stem cells (MSCs), which require relatively little oxygen. This is how the blood vessels are produced to provide oxygen to the osteocytes. Studies on carriers capable of accommodating recombinant human bone morphogenetic protein-2 (rhBMP-2) have continued and many substrates have been used. There is absorbable collagen sponge, hyaluronan, calcium phosphate, deproteinized bovine, bone matrix, hydroxyapatite (HA), polylactic acids, polymethylethyl-methacrylate of this kind (5,11,13,24). These should be similar to the structure and biological functions of the extracellular matrix as healing sites and should mechanically support space, transport induction molecules or cells to the beneficial areas, and regulate the structure and function of newly formed tissues.

In recent years, scaffold with excellent biocompatibility, proper mechanical properties, biodegradability and porosity have been in the spotlight. Other essential factors of the support should be immune and reproducible. It should help cells ingrown by enabling blood vessels to penetrate, and be biocompatible and biodegradable (2,20,22). It should also be a carrier of bone forming factors, which should have a proper secretion mechanism for growth factors and maintain the rate at which bone forming factors are secreted locally. Among them, hydrogel is a three-dimensional polymer network formed by hydrophilic polymer chain through physiological and chemical bonds. Hydrogel has the property of swelling a large amount of water inside when it is aqueous solution, and when it is, it consists of more than 90 percent water.

Polymeric hydrogel has the advantage of low surface tension due to the high fluidity of the macromolecule chain on the surface, which facilitates material transfer inside and outside the hydrogel, having similar flexibility to normal cell tissue with high moisture content, and good biocompatibility (6,8). Currently, for better functionality and utilization, biological materials can be used in combination to control physical and chemical properties, and hybrid polymer hydrogels with various functions are actively produced and applied (15). In this study, we wanted to see the effect of the bone formation in the calvarial defect model by using keratin-based hydrogel made with human hair as a scaffold. To see if the inherent abilities of BMP are well demonstrated when keratin-based hydrogel is used as scaffold in a group using BMP-2, we looked at the creation of blood vessels using bone formation using Micro-computed tomography (micro-CT), immunohistochemistry.

Experimental animals

The twelve C57BL/6 mice (6 weeks after the birth) were prepared for the study. Their weight are measured 25-30 g. The mice had acclimation period and quarantine period during 7 days. A standardized diet was implemented. The breeding and management of experimental animals and surgical procedures followed the animal testing standards guidelines of the Institutional Animal Care and Use Committees (IACUC) at Jeonbuk National University, South Korea (JBNU 2020-0160).

Implant materials

The rhBMP-2 used in this study is NOVOSIS (rhBMP-2 0.25 mg, Daewoong Pharmaceutical Company, Seoul, South Korea), and the collagen-membrane is GENOSS collagen membrane (Genoss, Suwon, South Korea). Hydrogel used in this study is discarded keratin-based biocomposite hydrogels. The author used the same method of the following previously published report (3,4,12). First, human hair was washed water containing 0.5% Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, MA, USA). Then, was rinsed with fresh water and air-dried. The author extracted keratin through sulfitolysis. We extracted the cleaned hair and wool separately with 20% ethanol and 60% acetone for half of day using a soxhlet apparatus. Soxhelet apparatus is used for removing external lipids and impurities. Then, we prepared 150 g of the dried hair and wool samples. Then cut this sample into fragments of several millimeters. Then this was placed into 1.5 L of an aqueous solution containing 8 M urea, 75 g SDS and 150 g Na2S2O5. The author heated the mixture to 100°C, smoothly stirred for 30 min, cooled in water box at 30°C. Then, the resulting mixture was filtered by a mesh, and the filtrate material was dialyzed against 15 L of water containing 0.1% (W/V) Na2S2O5. It placed in cellulose tube for 72 hours. Next, we changed the dialysis solution twice per day and produced sheet type hydrogels that contained keratin protein (120 mm × 120 mm × 5 mm, width × length × thickness) (18). To sum up, a S-sulfo keratin solution containing numerous weight fractions of S-sulfo keratin was mixed with the PVA solution. It improves the gelation of the S-sulfo keratin liquid solution. The optimal concentrations of the S-sulfo keratin and polyvinyl alcohol (PVA) solutions were 5.0% (w/v) and 5.0% (w/v) respectively, 0.01% (w/v) polyethylenimine (PEI) included in the PVA solution (18). Later, the author moved S-sulfo keratin + PVA solution to the square dishes. And the sample was irradiated with an electron-beam accelerator (EB Tech Co., Ltd., Korea). The accelerator conditions are as follows. Beam current was set 8.5 mA, beam energy was set 2.5 MeV, irradiation width was set 110 cm, dose rate was set 6.67 kGy/s, conveyor velocity was set 10 m/min, at a value of 10-100 kGy at laboratory temperature under atmospheric conditions (13,14). After gelation, the author cut the hydrogels into the required sizes for conducting various analyses.

Experimental group selection

The whole animal was set up in three groups. Collagen-membrane film (n = 4) was applied to bone defect (group 1). Only hydrogel film (n = 4) was applied to bone defect (group 2). Hydrogel + rhBMP-2 (n = 4) was applied to bone defect (group 3).

Surgical procedure

We anesthetized mice with 2,2,2 – Tribromoethanol (Avertin, Sigma-Aldrich, MA, USA) using IACUC approved procedures. Next, the author pinched tail to identify the animal is fully sedated. Using a clipper, we shaved the hair at the expected surgical site. Dexamethasone (0.2 mg/Kg, subcutaneous; JEIL Pharm Co., Daegu, Korea) and carprofen (5 mg/kg, subcutaneous; Zoetis Inc., NJ, USA) are administered to prevent brain edema or an inflammatory response. The skin over the top of the skull is removed. Then the author started with a horizontal cut along the base of the head almost reaching the eyelid. Periosteum was elevated in full-thickness. Following the procedure, we drilled for a circle type defect about 4 mm in diameter with dental drill (16).

After a slight drilling, we applied lidocaine (JEIL Pharm Co., Daegu, Korea), epinephrine (JEIL Pharm Co., Daegu, Korea) solution again onto the skull surface. The drilling was stopped when a very thin layer of bone is left. The bone fragment of this formed deficit was gently elevated with curette. We identified that the dura mater is not damaged and that there is no bleeding. Each implant was applied to the bone deficit (Fig. 1). Following the surgical procedure, the periosteum was sutured with absorbable suture material (4-0 Vicryl, reverse cutting 19 mm 3/8 C, Ethicon, Somerville, NJ, USA) first, then sutured the skin with 3-0 nylon (AILEE CO., LTD., Busan, Korea). The mice were received appropriate postoperative analgesia and antibiotics. After the 2 weeks, the suture was removed. During bone healing, no signs of infection were identified.

Figure 1.Gross appearance of each group immediately following surgery. Group 1 (A) applied only the collagen membrane to the bone defect, and the Hydrogel group (B) applied the keratin-based hydrogel to the bone defect. Hydrogel + BMP group (C) applied keratin-based hydrogel and BMP to the bone defect.

Micro-CT evaluation

Microradiography was performed using micro-CT scanner (Skyscan 1076, Bucker, Belgium). The author dropped normal saline to the samples for moistening during the micro-CT scanning to prevent the sample drying. The scanning, with a resolution of 35 μm, was performed with an energy of 100 kV and intensity of 100 μA. The data from micro-CT were reconstructed into Nrecon (SKYSCAN, Bucker, Belguim) programs. This was analyzed in three dimensions by CT-ANTM (SKYSCAN, Bucker, Belgium) program, CT-VolTM (SKYSCAN, Bucker, Belgium) program, and then measured the size of the bone defect (Tissue volume, [TV; mm3], and newly grown bones (Bone volume, [BV; mm3]. The ratio of newly grown bones to total bone defects was calculated by converting them into percentages (BV/TV; %).

Immunohistochemistry evaluation

For immunofluorescence studies, the skull of the mouse was fixed in 4% paraformaldehyde for 4 hours. Then the sample was dehydrated in 20% sucrose solution overnight. Then the sample was embedded in tissue freezing medium (Leica, Wetzlar, Germany). Then the sample was carried out with Whole mount staining (7). The author blocked the sectioned samples with 5% donkey serum in 0.3% Triton X-100 in phosphate buffered saline for 3 hours at room temperature. Then, the sample was incubated overnight at 4°C with the following primary antibodies: anti-CD31 (hamster, clone 2H8; Millipore Billerica, MA, USA), FITC-conjugated anti-alpha-smooth muscle actin (α-SMA) (mouse, clone 1A4; Sigma-Aldrich) (7). After several washes, the author incubated the samples for 2 hours at Room temperature with the following secondary antibodies: Cy3-conjugated donkey anti-rabbit IgG, Cy3-conjugated goat anti-Armenian hamster IgG, FITC-conjugated goat anti-Armenian hamster IgG, Cy3-conjugated donkey anti-mouse IgG, FITC-conjugated goat anti-Armenian hamster IgG (all from Jackson Immuno Research, West Grove, PA, USA) (7). The Nuclei was stained with 4’, 6-diamidino-2-phenylindole (7). The samples were then mounted in Fluorescent Mounting Medium (Dako, Carpinteria, CA, USA). And immunofluorescent images were acquired by using a laser-scanning confocal microscope (LSM 880 with Airyscan; Carl Zeiss, Jena, Germany) (7).

Statistical analysis

Sham (collagen-membrane), Keratin based hydrogel, rhBMP-2 + Keratin based hydrogel data were compared. Micro-CT data and IHC data were conducted with Kruskal-Wallis test. Post-hoc test was conducted with Mann-Whitney U test and Bonferroni correction. Statistical significance was set at p < 0.05. The data are expressed as the mean ± SD. Statistical analyses were conducted using SPSS version 26.0 (IBM, Armonk, NY, USA).

Experimental follow-up

All the mouse recovered well without any clinical signs of complications in 4 weeks.

Gross evaluation

The visual evaluation was compared with a bone defect (Fig. 2). The bone defect in group 1 was expressed as 100% and the value of bone defect in group 2 and group 3 was compared to group 1 and expressed as a percentage. It was statistically significant that there was a decrease in bone defects in group 2 and group 3 compared to group 1 (Fig. 3A).

Figure 2.Visual change after 4 weeks of surgery. The inside of the white dotted line indicates a bone defect. Sham (A), hydrogel (B), hydrogel + BMP (C). Bone defect of the skull calculated by micro-CT between groups after 4 weeks of surgery. The inside of the yellow dotted line indicates a bone defect. Sham (D), hydrogel (E), hydrogel + BMP (F).
Figure 3.Various factors between groups 4 weeks after surgery. Bone defect difference (A). Tissue volume calculating by micro-CT (B). Bone volume calculating by micro-CT (C). Bone volume/tissue volume calculating by micro-CT (D). Blood vessel density (E). α-SMA on CD31+ BVS (F). *p < 0.05, **p < 0.01.

Micro-CT evaluation

Tissue volume (TV) was measured in three groups. It was measured ranged from 10.14 to 10.63 mm3 in group 1, from 16.48 to 17.02 mm3 in group 2, from 23.31 to 25.01 mm3 in group 3. The mean TV of group 1 was 10.27 mm3. The mean TV of group 2 was 16.79 mm3. The mean TV of group 3 was 23.93 mm3 (Fig. 3B).

Bone volume (BV) was measured in three groups. It was measured ranged from 0.32 to 0.70 mm3 in group 1, from 1.87 to 2.13 mm3 in group 2, from 6.95 to 8.03 mm3 in group 3. The mean BV of group 1 was 0.50 mm3. The mean BV of group 2 was 2.06 mm3. The mean BV of group 3 was 7.73 mm3 (Fig. 3C).

Bone volume fraction was measured in three groups. It was measured ranged from 3.04 to 6.86% in group 1, from 11.35 to 12.6 % in group 2, from 27.79 to 34.4% in group 3. The mean bone volume fraction of group 1 was 4.86%. The mean bone volume fraction of group 2 was 12.27%. The mean bone volume fraction of group 3 was 32.36% (Fig. 3D).

It was statistically significant that there was a increase the new bone parameter in group 2 and group 3 compared to group 1.

Immunohistochemistry analysis

We compared the blood vessel density to each group and found that group 2, group 3 increased statistically compared to group 1 (Fig. 3E) (p < 0.05). Comparing a-SMA in CD31 showed that group 3 had a statistically significant increase over group 1 (Fig. 3F, 4).

Figure 4.Immunohistochemistry analysis was performed in each group. α-SMA is observed in hydrogel + BMP group.

The recovery of bone loss, which occurs after trauma or extensive resection, requires great effort to rebuild. The development of biotechnology techniques to promote the regeneration of bone tissue over the past few decades has been made by the use of various growth factors, osteogenic cells, and biocompatible scaffold. This field is actively being studied in both the fields of human medicine and veterinary medicine, and this author also wanted to examine the availability of materials in the bone defect model using a new biocompatible support system, one of these biotechnology techniques (17,18). BMP is known to be the most effective osteoplastic factor to induce the formation of new bone as a member of the transforming growth factor-β subfamily to recover the bone defect (25). Of these, BMP-2 is the most powerful osteoplastic facilitator and is characterized by playing an important role in many stages of the osteoplastic process. However, results are reported that excessive concentration of BMP promotes cell death and hinders bone formation (3,4,27).

Despite the excellent bone formation of BMP, the high cost and BMP easily melt within the tissues and spread to surrounding tissues. Therefore, considering that it only works in a short period of time and that high concentrations of BMP interfere with cell necrosis and bone formation, BMP requires the use of small amounts and rhBMP-2 carriers that can be released slowly. BMP generally used acellular collagen (ACS) made from Type 1 bovine collagen. Common use is a substance called INFUSE (Medtronic company, Minneapolis, USA). It consists of rhBMP-2, ACS substrate and diluent. The principle is that the collagen of the ACS has some binding BMP and the cells penetrate the ACS and are replaced by bone tissue (14). However, the ACS has 90 percent of its volume empty due to its low cross-link level, which makes it difficult to maintain its volume. Therefore, large bone defect such as this experiment are not recommended for bone regeneration.

The scaffold used in this study is a hydrogel made from which keratin is extracted using human hair and based on it. This material has already been published once in the development of hydrogel and once in the study of comparison of the healing effect of mouse wound (17,18). In treating the wound, the comparison with the hydrocolloid of this material proved to be excellent, which could be interpreted that the keratin-based hydrogel has excellent water retention ability to maintain water in the wound during treating the wound. The published paper on the implantation of BMP into mice in hydrogel with different moisture content showed that the retention period of BMP was sustained by the decrease in hydrogel moisture content (26). In addition, the alkaline phosphatase activity and osteocalcin content around the hydrogel was found to be higher than around the group injected only with BMP solutions or around the collagen sponge containing BMP (26).

Therefore, we looked into the possibility that the water retention effect of keratin-based hydrogel is excellent as a carrier of rhBMP that can increase the retention of rhBMP. In this experiment, we were able to see that group 2 and group 3 decreased compared to group 1 using collagen-membrane when visibly comparing the bone defect. In addition, on Mirco-CT, the bone volume, bone volume/tissue volume increased that group 2 and group 3 compared to group 1 and the statistical significance was also observed. It may be controversial to measure and judge the effect of bone regeneration using Mirco-CT only. However, a previously published report that the assessment of early bone formation during low-threshold shooting is no different from that of systematic measurement (21).

It is reliable that the bone volume has increased in that group 2 and group 3 using keratin-based hydrogel compared to group 1 that only processed collagen-membrane as a result of micro-CT. According to published other reports, ACP was added to the lower jaw of the adult dog as a scaffold for implantation, and rhBMP-2 of 0.2 mg/mL was added to the experimental group. Compared to controls that did not contain rhBMP, the experimental group containing BMP significantly formed many new bones, and the bone-to-implant contact percentage was also high (9). According to published other reports, implanted ACS containing rhBMP-2 in adult dogs, forming CSD bone loss. At this time, the concentration of rhBMP-2 was 0.05, 0.10 and 0.20 mg/mL, respectively, and the capacity of BMP was 200 g, 400 g and 800 g. After eight weeks, the deficit recovered 86%, 96% and 88%, respectively. However, there was no significant difference between these figures (23). Combining the above previous studies, most studies showed significant osteogenesis compared to control groups with only substrate implanted rhBMP. Similar results were obtained in this study.

Vascularization is also an important part of bone formation (19). It acts as a channel through which oxygen, nutrients, and factors such as parathyroid hormones, vitamin D, are supplied. Therefore, the vascularization process of the bone is one of the key processes in fracture healing. The authors investigated whether angiogenesis was enhanced within the hydrogel structure by immunohistochemical analysis of α-SMA, CD31, and DAPI, which are used as positive indices for angiogenesis. VEGF is cytokine produced by osteoblast and multiplies with endothelial cells, but was not performed in this experiment (12).

In this study, the blood vessel density increased statistically in group 2 and group 3 compared to group 1, but the blood vessel density between group 2 and group 3 was not much different. The cause of faster bone formation in group 2 and group 3 was found in terms of higher density of blood vessels, which further promoted movements of oxygen, nutrients, and factors such as parathyroid hormones, Vitamin D through blood. The evaluation of α-SMA showed that group 3 was significantly increased compared to group 1 and group 2. α-SMA is an indicator of the stabilization of blood vessels, characterized by CD-31 appearing later than the detection process. Therefore, it can be interpreted that blood vessels generated in group 3 with BMP are stabilized compared to other groups. In this way, immunohistochemistry suggests that BMP is a suitable support body to function properly, given that it is known as a substance that promotes the new formation of blood vessel. According to a previously published paper, hyaluronic acid (HA) was used as a scaffold for BMP-2 and human mesenchymal stem cells (hMSCs) for rat calvarial defect regeneration. Factors such as CD31 and VEFG did not appear in the BMP + hyaluronic acid base hydrogel group, which used only hyaluronic acid base hydrogel, and appeared in hMSCs + hyaluronic acid base hydrogel. In that study, it is believed that the stem cell present in the hydrogel secrete the recruiting signal of the vascular cell or endothelial progenitor cell when the cause is applied with the mesenchymal cell to the hydrogel (12).

It tends to see that the mesenchymal cell does not exist in hair, but rather a group of dead cells called keratin. The hydrogel used in this study is also based on keratin. In this study, although VEGF analysis was not performed, specific markers of vascular endothelial factors such as CD31 could be observed if only hydrogel was used and hydrogel + BMP was used. Based on this result, insert the mesenchymal cell into the hydrogel and carefully predict whether there is a difference in the vascular generation of BMP due to the difference in hydrogel rather than the stimulation of the stem cell. Therefore, keratin-based hydrogel is economical and environmentally friendly and can be a meaningful material in the field of tissue engineering in veterinary medicine by conducting further research in dogs and cats based on this study.

The limitation of this study is that there are only four mice in each group, and the follow-up period is only four weeks. In addition, with more mice and long-term follow-up studies, overall research is recommended for this material. Also, since there is no group treated with rhBMP-2 alone, it is difficult to make a more objective comparison between groups.

It was observed that the existing properties of rhBMP-2 were fully expressed when keratin-based hydrogel was used as Scaffold in calvarial skull defect mouse models that could not be treated with normal physiological reactions. Through gross evaluation, micro-CT, and immunohistochemistry, we can confirm that keratin-based hydrogel with rhBMP-2 has therapeutic effects in calvarial bone defects mouse model.

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Article

Original Article

J Vet Clin 2022; 39(6): 302-310

Published online December 31, 2022 https://doi.org/10.17555/jvc.2022.39.6.302

Copyright © The Korean Society of Veterinary Clinics.

Effect of Keratin-Based Biocomposite Hydrogels as a RhBMP-2 Carrier in Calvarial Bone Defects Mouse Model

Jongjin Lee , Jinsu Kang , Jaewon Seol , Namsoo Kim , Suyoung Heo

College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, Korea

Correspondence to:*syheo@jbnu.ac.kr

Received: August 12, 2022; Revised: October 14, 2022; Accepted: November 23, 2022

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Recently, in human medicine and veterinary medicine, interest in synthetic bone graft is increasing. Among them, bone morphogenic protein (BMP) is currently being actively researched and applied to clinical trials. However, BMP has the disadvantage of being expensive and easily absorbed into surrounding tissues. Therefore, BMP requires the use of small amounts and rhBMP (recombinant human bone morphogenetic protein)-2 carriers that can be released slowly. Hydrogel has the property of swelling a large amount of water inside when it is aqueous solution, and when it is, it consists of more than 90 percent water. Using these properties, hydrogels are often used as rhBMP-2 carrier. The scaffold used in this study is a hydrogel made from which keratin is extracted using human hair and based on it. In this study, we wanted to see the effect of bone formation in the calvarial defect model by using keratin-based hydrogel made with human hair as a scaffold. The experiment was conducted by dividing 3 groups a total of 12 mice. Calvarial bone defect is set to all 4 mm diameters. Bone formation was evaluated by using gross evaluation, micro-computed tomography (micro-CT), immunohistochemistry. Groups using keratin-based hydrogel were significantly observed compared to Group 1s, and the most bone formations were found when rhBMP-2 and hydrogel were used. This represents the superiority of the functions of the rhBMP-2 carrier by a new material, keratin-based hydrogel. Through gross evaluation, micro-CT, and immunohistochemistry, we can confirm that keratin-based hydrogel is a useful rhBMP-2 carrier.

Keywords: keratin, hydrogel, rhBMP-2 carrier, calvarial defect model, mouse.

Introduction

In the field of orthopedics and neurosurgery, bone transplants are required in many cases. Bone transplants are required in the reconstruction of congenital deficits and acquired lesions, or deficits caused by trauma. In addition, bone transplants are often essential in the number of dental fields in patients with severe bone absorption following the onset of chronic periodontitis. Forming a new bone is the domain of tissue engineering. Three major points of tissue engineering are the source of cells, matrix or scaffold, and signal moles. In terms of bone regeneration, this can be seen as osteoplastic cells, hormones with bone-inducing effects, growth factors, and substrate with bone conduction effects.

A lot of progress in the field of veterinary medicine is autograft, which is considered gold standard. However, it tends to be easy absorbed, and if a small dog’s bone graft is carried out, a fracture, a small amount of bone sample is sometimes a problem. Xenograft bone transplants that use tissues from different species have problems with immune response. Therefore, the field where much research has been conducted in human medicine and veterinary medicine recently is according to synthetic bone transplantation. This has the advantage of not having to perform any additional surgery on the other side of the body and being able to transplant to the extent planned by the surgeon. There is platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), bone morphogenetic proteins (BMP)-2, BMP-7 and insulin-like growth factors (IGF) I, II of this kind (1,20).

Among them, BMP is currently being actively researched and applied to clinical trials. BMP is involved in osteogenesis and chondrogenesis and is also involved in embryonic development and fracture healing. The mechanism at the cell level works in binding with the receptor on the surface of the cell. When BMP is coupled to a receptor, cascade through the serine/threonine kinase is expressed in the cell. After this process, the differential hepatocytes are differentiated into osteoporosis. It also allows the production of vascular endothelial group factor-α (VEFG-α) from osteoporosis to help produce angiogenesis essential to bone formation (10).

Osteocytes consume more oxygen than mesenchymal stem cells (MSCs), which require relatively little oxygen. This is how the blood vessels are produced to provide oxygen to the osteocytes. Studies on carriers capable of accommodating recombinant human bone morphogenetic protein-2 (rhBMP-2) have continued and many substrates have been used. There is absorbable collagen sponge, hyaluronan, calcium phosphate, deproteinized bovine, bone matrix, hydroxyapatite (HA), polylactic acids, polymethylethyl-methacrylate of this kind (5,11,13,24). These should be similar to the structure and biological functions of the extracellular matrix as healing sites and should mechanically support space, transport induction molecules or cells to the beneficial areas, and regulate the structure and function of newly formed tissues.

In recent years, scaffold with excellent biocompatibility, proper mechanical properties, biodegradability and porosity have been in the spotlight. Other essential factors of the support should be immune and reproducible. It should help cells ingrown by enabling blood vessels to penetrate, and be biocompatible and biodegradable (2,20,22). It should also be a carrier of bone forming factors, which should have a proper secretion mechanism for growth factors and maintain the rate at which bone forming factors are secreted locally. Among them, hydrogel is a three-dimensional polymer network formed by hydrophilic polymer chain through physiological and chemical bonds. Hydrogel has the property of swelling a large amount of water inside when it is aqueous solution, and when it is, it consists of more than 90 percent water.

Polymeric hydrogel has the advantage of low surface tension due to the high fluidity of the macromolecule chain on the surface, which facilitates material transfer inside and outside the hydrogel, having similar flexibility to normal cell tissue with high moisture content, and good biocompatibility (6,8). Currently, for better functionality and utilization, biological materials can be used in combination to control physical and chemical properties, and hybrid polymer hydrogels with various functions are actively produced and applied (15). In this study, we wanted to see the effect of the bone formation in the calvarial defect model by using keratin-based hydrogel made with human hair as a scaffold. To see if the inherent abilities of BMP are well demonstrated when keratin-based hydrogel is used as scaffold in a group using BMP-2, we looked at the creation of blood vessels using bone formation using Micro-computed tomography (micro-CT), immunohistochemistry.

Materials and Methods

Experimental animals

The twelve C57BL/6 mice (6 weeks after the birth) were prepared for the study. Their weight are measured 25-30 g. The mice had acclimation period and quarantine period during 7 days. A standardized diet was implemented. The breeding and management of experimental animals and surgical procedures followed the animal testing standards guidelines of the Institutional Animal Care and Use Committees (IACUC) at Jeonbuk National University, South Korea (JBNU 2020-0160).

Implant materials

The rhBMP-2 used in this study is NOVOSIS (rhBMP-2 0.25 mg, Daewoong Pharmaceutical Company, Seoul, South Korea), and the collagen-membrane is GENOSS collagen membrane (Genoss, Suwon, South Korea). Hydrogel used in this study is discarded keratin-based biocomposite hydrogels. The author used the same method of the following previously published report (3,4,12). First, human hair was washed water containing 0.5% Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, MA, USA). Then, was rinsed with fresh water and air-dried. The author extracted keratin through sulfitolysis. We extracted the cleaned hair and wool separately with 20% ethanol and 60% acetone for half of day using a soxhlet apparatus. Soxhelet apparatus is used for removing external lipids and impurities. Then, we prepared 150 g of the dried hair and wool samples. Then cut this sample into fragments of several millimeters. Then this was placed into 1.5 L of an aqueous solution containing 8 M urea, 75 g SDS and 150 g Na2S2O5. The author heated the mixture to 100°C, smoothly stirred for 30 min, cooled in water box at 30°C. Then, the resulting mixture was filtered by a mesh, and the filtrate material was dialyzed against 15 L of water containing 0.1% (W/V) Na2S2O5. It placed in cellulose tube for 72 hours. Next, we changed the dialysis solution twice per day and produced sheet type hydrogels that contained keratin protein (120 mm × 120 mm × 5 mm, width × length × thickness) (18). To sum up, a S-sulfo keratin solution containing numerous weight fractions of S-sulfo keratin was mixed with the PVA solution. It improves the gelation of the S-sulfo keratin liquid solution. The optimal concentrations of the S-sulfo keratin and polyvinyl alcohol (PVA) solutions were 5.0% (w/v) and 5.0% (w/v) respectively, 0.01% (w/v) polyethylenimine (PEI) included in the PVA solution (18). Later, the author moved S-sulfo keratin + PVA solution to the square dishes. And the sample was irradiated with an electron-beam accelerator (EB Tech Co., Ltd., Korea). The accelerator conditions are as follows. Beam current was set 8.5 mA, beam energy was set 2.5 MeV, irradiation width was set 110 cm, dose rate was set 6.67 kGy/s, conveyor velocity was set 10 m/min, at a value of 10-100 kGy at laboratory temperature under atmospheric conditions (13,14). After gelation, the author cut the hydrogels into the required sizes for conducting various analyses.

Experimental group selection

The whole animal was set up in three groups. Collagen-membrane film (n = 4) was applied to bone defect (group 1). Only hydrogel film (n = 4) was applied to bone defect (group 2). Hydrogel + rhBMP-2 (n = 4) was applied to bone defect (group 3).

Surgical procedure

We anesthetized mice with 2,2,2 – Tribromoethanol (Avertin, Sigma-Aldrich, MA, USA) using IACUC approved procedures. Next, the author pinched tail to identify the animal is fully sedated. Using a clipper, we shaved the hair at the expected surgical site. Dexamethasone (0.2 mg/Kg, subcutaneous; JEIL Pharm Co., Daegu, Korea) and carprofen (5 mg/kg, subcutaneous; Zoetis Inc., NJ, USA) are administered to prevent brain edema or an inflammatory response. The skin over the top of the skull is removed. Then the author started with a horizontal cut along the base of the head almost reaching the eyelid. Periosteum was elevated in full-thickness. Following the procedure, we drilled for a circle type defect about 4 mm in diameter with dental drill (16).

After a slight drilling, we applied lidocaine (JEIL Pharm Co., Daegu, Korea), epinephrine (JEIL Pharm Co., Daegu, Korea) solution again onto the skull surface. The drilling was stopped when a very thin layer of bone is left. The bone fragment of this formed deficit was gently elevated with curette. We identified that the dura mater is not damaged and that there is no bleeding. Each implant was applied to the bone deficit (Fig. 1). Following the surgical procedure, the periosteum was sutured with absorbable suture material (4-0 Vicryl, reverse cutting 19 mm 3/8 C, Ethicon, Somerville, NJ, USA) first, then sutured the skin with 3-0 nylon (AILEE CO., LTD., Busan, Korea). The mice were received appropriate postoperative analgesia and antibiotics. After the 2 weeks, the suture was removed. During bone healing, no signs of infection were identified.

Figure 1. Gross appearance of each group immediately following surgery. Group 1 (A) applied only the collagen membrane to the bone defect, and the Hydrogel group (B) applied the keratin-based hydrogel to the bone defect. Hydrogel + BMP group (C) applied keratin-based hydrogel and BMP to the bone defect.

Micro-CT evaluation

Microradiography was performed using micro-CT scanner (Skyscan 1076, Bucker, Belgium). The author dropped normal saline to the samples for moistening during the micro-CT scanning to prevent the sample drying. The scanning, with a resolution of 35 μm, was performed with an energy of 100 kV and intensity of 100 μA. The data from micro-CT were reconstructed into Nrecon (SKYSCAN, Bucker, Belguim) programs. This was analyzed in three dimensions by CT-ANTM (SKYSCAN, Bucker, Belgium) program, CT-VolTM (SKYSCAN, Bucker, Belgium) program, and then measured the size of the bone defect (Tissue volume, [TV; mm3], and newly grown bones (Bone volume, [BV; mm3]. The ratio of newly grown bones to total bone defects was calculated by converting them into percentages (BV/TV; %).

Immunohistochemistry evaluation

For immunofluorescence studies, the skull of the mouse was fixed in 4% paraformaldehyde for 4 hours. Then the sample was dehydrated in 20% sucrose solution overnight. Then the sample was embedded in tissue freezing medium (Leica, Wetzlar, Germany). Then the sample was carried out with Whole mount staining (7). The author blocked the sectioned samples with 5% donkey serum in 0.3% Triton X-100 in phosphate buffered saline for 3 hours at room temperature. Then, the sample was incubated overnight at 4°C with the following primary antibodies: anti-CD31 (hamster, clone 2H8; Millipore Billerica, MA, USA), FITC-conjugated anti-alpha-smooth muscle actin (α-SMA) (mouse, clone 1A4; Sigma-Aldrich) (7). After several washes, the author incubated the samples for 2 hours at Room temperature with the following secondary antibodies: Cy3-conjugated donkey anti-rabbit IgG, Cy3-conjugated goat anti-Armenian hamster IgG, FITC-conjugated goat anti-Armenian hamster IgG, Cy3-conjugated donkey anti-mouse IgG, FITC-conjugated goat anti-Armenian hamster IgG (all from Jackson Immuno Research, West Grove, PA, USA) (7). The Nuclei was stained with 4’, 6-diamidino-2-phenylindole (7). The samples were then mounted in Fluorescent Mounting Medium (Dako, Carpinteria, CA, USA). And immunofluorescent images were acquired by using a laser-scanning confocal microscope (LSM 880 with Airyscan; Carl Zeiss, Jena, Germany) (7).

Statistical analysis

Sham (collagen-membrane), Keratin based hydrogel, rhBMP-2 + Keratin based hydrogel data were compared. Micro-CT data and IHC data were conducted with Kruskal-Wallis test. Post-hoc test was conducted with Mann-Whitney U test and Bonferroni correction. Statistical significance was set at p < 0.05. The data are expressed as the mean ± SD. Statistical analyses were conducted using SPSS version 26.0 (IBM, Armonk, NY, USA).

Results

Experimental follow-up

All the mouse recovered well without any clinical signs of complications in 4 weeks.

Gross evaluation

The visual evaluation was compared with a bone defect (Fig. 2). The bone defect in group 1 was expressed as 100% and the value of bone defect in group 2 and group 3 was compared to group 1 and expressed as a percentage. It was statistically significant that there was a decrease in bone defects in group 2 and group 3 compared to group 1 (Fig. 3A).

Figure 2. Visual change after 4 weeks of surgery. The inside of the white dotted line indicates a bone defect. Sham (A), hydrogel (B), hydrogel + BMP (C). Bone defect of the skull calculated by micro-CT between groups after 4 weeks of surgery. The inside of the yellow dotted line indicates a bone defect. Sham (D), hydrogel (E), hydrogel + BMP (F).
Figure 3. Various factors between groups 4 weeks after surgery. Bone defect difference (A). Tissue volume calculating by micro-CT (B). Bone volume calculating by micro-CT (C). Bone volume/tissue volume calculating by micro-CT (D). Blood vessel density (E). α-SMA on CD31+ BVS (F). *p < 0.05, **p < 0.01.

Micro-CT evaluation

Tissue volume (TV) was measured in three groups. It was measured ranged from 10.14 to 10.63 mm3 in group 1, from 16.48 to 17.02 mm3 in group 2, from 23.31 to 25.01 mm3 in group 3. The mean TV of group 1 was 10.27 mm3. The mean TV of group 2 was 16.79 mm3. The mean TV of group 3 was 23.93 mm3 (Fig. 3B).

Bone volume (BV) was measured in three groups. It was measured ranged from 0.32 to 0.70 mm3 in group 1, from 1.87 to 2.13 mm3 in group 2, from 6.95 to 8.03 mm3 in group 3. The mean BV of group 1 was 0.50 mm3. The mean BV of group 2 was 2.06 mm3. The mean BV of group 3 was 7.73 mm3 (Fig. 3C).

Bone volume fraction was measured in three groups. It was measured ranged from 3.04 to 6.86% in group 1, from 11.35 to 12.6 % in group 2, from 27.79 to 34.4% in group 3. The mean bone volume fraction of group 1 was 4.86%. The mean bone volume fraction of group 2 was 12.27%. The mean bone volume fraction of group 3 was 32.36% (Fig. 3D).

It was statistically significant that there was a increase the new bone parameter in group 2 and group 3 compared to group 1.

Immunohistochemistry analysis

We compared the blood vessel density to each group and found that group 2, group 3 increased statistically compared to group 1 (Fig. 3E) (p < 0.05). Comparing a-SMA in CD31 showed that group 3 had a statistically significant increase over group 1 (Fig. 3F, 4).

Figure 4. Immunohistochemistry analysis was performed in each group. α-SMA is observed in hydrogel + BMP group.

Discussion

The recovery of bone loss, which occurs after trauma or extensive resection, requires great effort to rebuild. The development of biotechnology techniques to promote the regeneration of bone tissue over the past few decades has been made by the use of various growth factors, osteogenic cells, and biocompatible scaffold. This field is actively being studied in both the fields of human medicine and veterinary medicine, and this author also wanted to examine the availability of materials in the bone defect model using a new biocompatible support system, one of these biotechnology techniques (17,18). BMP is known to be the most effective osteoplastic factor to induce the formation of new bone as a member of the transforming growth factor-β subfamily to recover the bone defect (25). Of these, BMP-2 is the most powerful osteoplastic facilitator and is characterized by playing an important role in many stages of the osteoplastic process. However, results are reported that excessive concentration of BMP promotes cell death and hinders bone formation (3,4,27).

Despite the excellent bone formation of BMP, the high cost and BMP easily melt within the tissues and spread to surrounding tissues. Therefore, considering that it only works in a short period of time and that high concentrations of BMP interfere with cell necrosis and bone formation, BMP requires the use of small amounts and rhBMP-2 carriers that can be released slowly. BMP generally used acellular collagen (ACS) made from Type 1 bovine collagen. Common use is a substance called INFUSE (Medtronic company, Minneapolis, USA). It consists of rhBMP-2, ACS substrate and diluent. The principle is that the collagen of the ACS has some binding BMP and the cells penetrate the ACS and are replaced by bone tissue (14). However, the ACS has 90 percent of its volume empty due to its low cross-link level, which makes it difficult to maintain its volume. Therefore, large bone defect such as this experiment are not recommended for bone regeneration.

The scaffold used in this study is a hydrogel made from which keratin is extracted using human hair and based on it. This material has already been published once in the development of hydrogel and once in the study of comparison of the healing effect of mouse wound (17,18). In treating the wound, the comparison with the hydrocolloid of this material proved to be excellent, which could be interpreted that the keratin-based hydrogel has excellent water retention ability to maintain water in the wound during treating the wound. The published paper on the implantation of BMP into mice in hydrogel with different moisture content showed that the retention period of BMP was sustained by the decrease in hydrogel moisture content (26). In addition, the alkaline phosphatase activity and osteocalcin content around the hydrogel was found to be higher than around the group injected only with BMP solutions or around the collagen sponge containing BMP (26).

Therefore, we looked into the possibility that the water retention effect of keratin-based hydrogel is excellent as a carrier of rhBMP that can increase the retention of rhBMP. In this experiment, we were able to see that group 2 and group 3 decreased compared to group 1 using collagen-membrane when visibly comparing the bone defect. In addition, on Mirco-CT, the bone volume, bone volume/tissue volume increased that group 2 and group 3 compared to group 1 and the statistical significance was also observed. It may be controversial to measure and judge the effect of bone regeneration using Mirco-CT only. However, a previously published report that the assessment of early bone formation during low-threshold shooting is no different from that of systematic measurement (21).

It is reliable that the bone volume has increased in that group 2 and group 3 using keratin-based hydrogel compared to group 1 that only processed collagen-membrane as a result of micro-CT. According to published other reports, ACP was added to the lower jaw of the adult dog as a scaffold for implantation, and rhBMP-2 of 0.2 mg/mL was added to the experimental group. Compared to controls that did not contain rhBMP, the experimental group containing BMP significantly formed many new bones, and the bone-to-implant contact percentage was also high (9). According to published other reports, implanted ACS containing rhBMP-2 in adult dogs, forming CSD bone loss. At this time, the concentration of rhBMP-2 was 0.05, 0.10 and 0.20 mg/mL, respectively, and the capacity of BMP was 200 g, 400 g and 800 g. After eight weeks, the deficit recovered 86%, 96% and 88%, respectively. However, there was no significant difference between these figures (23). Combining the above previous studies, most studies showed significant osteogenesis compared to control groups with only substrate implanted rhBMP. Similar results were obtained in this study.

Vascularization is also an important part of bone formation (19). It acts as a channel through which oxygen, nutrients, and factors such as parathyroid hormones, vitamin D, are supplied. Therefore, the vascularization process of the bone is one of the key processes in fracture healing. The authors investigated whether angiogenesis was enhanced within the hydrogel structure by immunohistochemical analysis of α-SMA, CD31, and DAPI, which are used as positive indices for angiogenesis. VEGF is cytokine produced by osteoblast and multiplies with endothelial cells, but was not performed in this experiment (12).

In this study, the blood vessel density increased statistically in group 2 and group 3 compared to group 1, but the blood vessel density between group 2 and group 3 was not much different. The cause of faster bone formation in group 2 and group 3 was found in terms of higher density of blood vessels, which further promoted movements of oxygen, nutrients, and factors such as parathyroid hormones, Vitamin D through blood. The evaluation of α-SMA showed that group 3 was significantly increased compared to group 1 and group 2. α-SMA is an indicator of the stabilization of blood vessels, characterized by CD-31 appearing later than the detection process. Therefore, it can be interpreted that blood vessels generated in group 3 with BMP are stabilized compared to other groups. In this way, immunohistochemistry suggests that BMP is a suitable support body to function properly, given that it is known as a substance that promotes the new formation of blood vessel. According to a previously published paper, hyaluronic acid (HA) was used as a scaffold for BMP-2 and human mesenchymal stem cells (hMSCs) for rat calvarial defect regeneration. Factors such as CD31 and VEFG did not appear in the BMP + hyaluronic acid base hydrogel group, which used only hyaluronic acid base hydrogel, and appeared in hMSCs + hyaluronic acid base hydrogel. In that study, it is believed that the stem cell present in the hydrogel secrete the recruiting signal of the vascular cell or endothelial progenitor cell when the cause is applied with the mesenchymal cell to the hydrogel (12).

It tends to see that the mesenchymal cell does not exist in hair, but rather a group of dead cells called keratin. The hydrogel used in this study is also based on keratin. In this study, although VEGF analysis was not performed, specific markers of vascular endothelial factors such as CD31 could be observed if only hydrogel was used and hydrogel + BMP was used. Based on this result, insert the mesenchymal cell into the hydrogel and carefully predict whether there is a difference in the vascular generation of BMP due to the difference in hydrogel rather than the stimulation of the stem cell. Therefore, keratin-based hydrogel is economical and environmentally friendly and can be a meaningful material in the field of tissue engineering in veterinary medicine by conducting further research in dogs and cats based on this study.

The limitation of this study is that there are only four mice in each group, and the follow-up period is only four weeks. In addition, with more mice and long-term follow-up studies, overall research is recommended for this material. Also, since there is no group treated with rhBMP-2 alone, it is difficult to make a more objective comparison between groups.

Conclusions

It was observed that the existing properties of rhBMP-2 were fully expressed when keratin-based hydrogel was used as Scaffold in calvarial skull defect mouse models that could not be treated with normal physiological reactions. Through gross evaluation, micro-CT, and immunohistochemistry, we can confirm that keratin-based hydrogel with rhBMP-2 has therapeutic effects in calvarial bone defects mouse model.

Conflicts of Interest

The authors have no conflicting interests.

Fig 1.

Figure 1.Gross appearance of each group immediately following surgery. Group 1 (A) applied only the collagen membrane to the bone defect, and the Hydrogel group (B) applied the keratin-based hydrogel to the bone defect. Hydrogel + BMP group (C) applied keratin-based hydrogel and BMP to the bone defect.
Journal of Veterinary Clinics 2022; 39: 302-310https://doi.org/10.17555/jvc.2022.39.6.302

Fig 2.

Figure 2.Visual change after 4 weeks of surgery. The inside of the white dotted line indicates a bone defect. Sham (A), hydrogel (B), hydrogel + BMP (C). Bone defect of the skull calculated by micro-CT between groups after 4 weeks of surgery. The inside of the yellow dotted line indicates a bone defect. Sham (D), hydrogel (E), hydrogel + BMP (F).
Journal of Veterinary Clinics 2022; 39: 302-310https://doi.org/10.17555/jvc.2022.39.6.302

Fig 3.

Figure 3.Various factors between groups 4 weeks after surgery. Bone defect difference (A). Tissue volume calculating by micro-CT (B). Bone volume calculating by micro-CT (C). Bone volume/tissue volume calculating by micro-CT (D). Blood vessel density (E). α-SMA on CD31+ BVS (F). *p < 0.05, **p < 0.01.
Journal of Veterinary Clinics 2022; 39: 302-310https://doi.org/10.17555/jvc.2022.39.6.302

Fig 4.

Figure 4.Immunohistochemistry analysis was performed in each group. α-SMA is observed in hydrogel + BMP group.
Journal of Veterinary Clinics 2022; 39: 302-310https://doi.org/10.17555/jvc.2022.39.6.302

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Vol.39 No.6 2022-12-31

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