Morphological and biomechanical effects of vitamin K2 on fracture healing: An animal study on the rat tibia fracture model

Abstract

Objective:

The aim of this study was to evaluate the effects of vitamin K2 on fracture healing.

Methods:

Twenty-four 6-week-old male Wistar albino rats that had open tibia fractures induced were included in this study. They were divided into 2 groups of 12, a group that had vitamin K2 administered over 30 consecutive days and a control group. After 30 days, the rats were sacrificed, and from each group, 6 tibiae were selected for biomechanical testing to examine the mechanical strength of the callus tissue using the Instron 3-point bending test and 6 tibiae were selected for histological analysis to examine the density and organization of callus tissue using Allen’s grading system and Huo et al’s grading system. Furthermore, weekly x-rays were taken to evaluate bone union described by Lane and Sandhu, and osteocalcin, procollagen I N-terminal propeptide, and procollagen I C-terminal propeptide were examined in blood samples taken by intracardiac puncture during sacrification.

Results:

Breaking force (P = .047), breaking time (P = .019), stiffness (P = .039), fracture strength (P = .041), and Young’s modulus (P = .032) showed a statistically significant increase in the K2 group. Procollagen I C-terminal propeptide (P = .024), procollagen I N-terminal propeptide (.047), and osteocalcin (.048) levels were significantly higher in the K2 group compared to the control group. Furthermore, 3rd-week x-rays showed higher bone union scores according to the Lane and Sandhu method in the K2 group (P = .014). However, the histological grading systems of Allen and Huo et al did not show statistically significant differences between groups (P = .086, P = .07, respectively).

Conclusion:

In light of these findings, it could be concluded that vitamin K2 has a significant positive effect on fracture healing.

HIGHLIGHTS

• Involved in many carboxylation reactions, Vitamin K is an important fat-­soluble vitamin for the bone metabolism. This study aimed to examine the effects of vitamin K2 on the fracture healing process.

• The study results showed that after administering vitamin K2 for 30 consecutive days in an open tibia fracture model on rats, there was a significant increase in breaking force, time, stiffness, fracture strength, and Young’s modulus.

• The results from this study suggests that Vitamin K2 has a beneficial impact on fracture healing by promoting the formation of a more durable bone callus.

Introduction

Vitamin K is a fat-soluble vitamin necessary for the function of many proteins involved in blood clotting.¹ Vitamin K is the name of a group consisting of phylloquinone (K1), menaquinone (K2), and menadione (K3) structures that are similar in network, fat-soluble 2-methyl-1,4 naphthoquinone.² Vitamin K functions as a cofactor in many biochemical pathways. The most important of these are vitamin K-dependent carboxylation reactions. In these reactions, the reducing form of vitamin K (quinol) transforms glutamate (Glu) into γ-carboxyglutamate (Gla) by γ-glutamyl carboxylase enzyme, while it transforms itself into epoxide (quinone) form, this is an energy-requiring reaction.³

There are 3 important bone-matrix proteins (osteocalcin, matrix Gla protein, and protein S) that are known to be dependent on vitamin K for bone formation.⁴ The carboxyglutamate residue is required for the biological activity of proteins such as calbindin and osteocalcin. With vitamin K-dependent carboxylation of osteocalcin, γ-carboxyglutamic acid (Gla) residues are formed from glutamate residues at positions 17, 21, and 24. This modification causes conformational change; the protein's α-annular structures become more stable. This structure provides high affinity for calcium and hydroxyapatite. In addition to γ-carboxylation of osteocalcin, vitamin K also affects other parameters of bone metabolism such as calcium homeostasis, prostaglandin E2, and interleukin-6 production.⁵ Studies examining the effects of vitamin K2 on bone metabolism found a significant increase in bone turnover markers (such as procollagen I C-terminal propeptide (PICP), procollagen I N-terminal propeptide (PINP), and osteocalcin) after vitamin K2 treatment. Procollagen I N-terminal propeptide consists of type I collagen synthesized by osteoblasts in bone structure. Therefore, they are often used for quantitative measurements of newly formed type I collagen. With a proliferation in osteoblasts and fibroblasts, there is a specific increase in PICP, so it is a direct marker of bone formation.⁶ Osteocalcin is used as an important marker in bone turnover and is produced by osteoblasts during bone formation. It is also a vitamin K-dependent protein that affects the synthesis and regulation of the bone matrix.

From this point on, in this study, we designed an animal experiment with rats and aimed to examine the effects of vitamin K2 on the fracture healing process. The following hypotheses have been tested: (1) mechanical strength of the callus of K2-treated rats was higher than that of untreated rats; (2) There are differences in histological features of callus tissue between K2 and control groups; (3) In blood analysis, some specific osteogenic markers were higher in rats given K2 than those not given.

Materials and Methods

Study design and animals

Based on our pre-study power analysis, group sizes of 12 would be enough to detect a difference of 27% difference between groups. Six-week-old 24 male Wistar albino rats were used. Rats were divided into 2 groups with simple randomization: control (n = 12) group and K2 (vitamin K2 given) (n = 12) group. After surgical open fracture model⁷ was applied, the rats were housed individually in stainless steel cages under controlled conditions: temperature of 17 -27°C, relative humidity of 35%-76%, a minimum of 5 air changes per hour, and a 12-h light and 12-h dark cycle. The animals were fed standard rat chow and municipal water (using water bottles) ad libitum.

Surgical procedures

The open tibia fractures were produced according to open fracture model.⁷ Anesthesia was applied with intraperitoneal injection of ketamine (20 mg/kg) and xylazine (5 mg/kg). Middle third of the right tibia was exposed by 2 cm anterior incision. The tissue around the tibia was bluntly dissected with hemostats. Apex of the tibial bow was identified, and blunt hemostat was passed just posterior to the tibia diaphysis to protect soft tissue. Osteotomy was made by gigli saw (Figure 1).

Figure 1.

Open fracture model of rat tibia.

17-G cannula needle was inserted through the fracture line proximally into medullary canal. Distal part of the needle was inserted distally through fracture line distally into medullary canal. After fixation of the fracture, surgery site was irrigated with saline solution and wound was closed with absorbable suture. After postoperative 1 day housing in the intensive care unit, all rats were followed in individual cages and not restricted from movement or feeding. The postoperative analgesia was provided by acetaminophen (160 mg/5 mL) drinking water for 3 weeks after surgery. After surgery, lateral x-ray was taken for confirmation of fracture fixation, and healing was followed by lateral x-ray of osteotomized tibia at days 7, 14, 21, and 30. After 4 weeks, all rats were sacrificed with cardiac puncture and blood draw.

Vitamin K2 administration

Synthetic menaquinone-7 (MK-7) (molecular formula: C₄₆H₆₄O₂) was provided by Phytonet AS, Istanbul, Turkey (manufactured by Kappa Bioscience AS, Oslo, Norway) and designated as KB-MK-7 (formerly SynMK-7).

Dosing formulations, consisting of a stock solution of 2.0 mg/mL MK-7 in corn oil, were prepared every 3 days. Animals were administered MK-7 at a dose of 5.0 mg/kg body weight/day by oral gavage for 30 consecutive days.⁸ After surgical procedure, MK7 application was initiated in the K2 group on the first postoperative day. Prior to dosing, MK-7 dosage formulations and corn oil were continuously mixed on a stir plate, protected from light, and aspirated into syringe for administration. MK-7 was administered by oral stainless-steel gavage needle. The same amount of corn oil was given by oral gavage to the control group each time to meet the same conditions.

Radiologic evaluation

Standard lateral radiographs (model E7339X, Toshiba Xray Rotanode, Otawara-shi, Japan) of the potted samples were taken using a digital cassette and processor (model CR-IR 392, FCR Prima T2 image reader; FujiFilm Corporation, Tokyo, Japan). Radiographs of each tibia were taken on days 0, 7, 14, 21, and 30 (after sacrifice) for following fracture healing stages. Radiological evaluation of the fracture healing was performed according to the methods of Lane and Sandhu⁹ (Table 1).

Table 1.

Radiologic evaluation criteria

Radiologic sign	Number
No healing	0
Callus Formation	1
Starting fractured bone healing	2
Disappearing of fracture line	3
Finished fractured bone healing	4

Blood analysis

At the end of 30 days, sacrification was performed with cardiac puncture and blood tests were performed on the blood samples taken during this procedure. Blood samples were taken in yellow-capped tubes (BD Diagnostics) and centrifuged for 10 minutes at 3000 g to obtain serum. Serum samples were stored at −80°C until measurement. In the blood samples, osteocalcin (Elabscience, Houston, Texas, US), PINP (Elabscience), and PICP (Elabscience) were measured by enzyme-linked immunosorbent assay (ELISA). Readings were made with a microplate reader (Biotek Synergy Reader).

Biomechanical evaluation

Six osteotomized tibia from each group were used for Instron 3-point bending test. All the osteotomized tibia were tested by conducting an Instron 3-point bending test to measure breaking force, breaking displacement, breaking time, stiffness, fracture strength, and Young’s modulus. Samples were placed between 3 supports according to the length of the tibia. Three-point bending strength was adjusted with a 30 mm length interval between the support spans. Loading span was placed in the middle of the support point. Loading span and 2 support spans were round to prevent shear load and damage to samples. The samples were placed horizontally with anterior surface upward position. Force was directed perpendicular to the center of the tibia diaphysis. Samples were loaded at 1 mm/sec speed until mechanical failure (Figure 2).

Figure 2.

Three-point bending test of the rat’s tibia.

Breaking force at failure was accepted as bending force. The slope of the linear part of the load–displacement curve was used to calculate stiffness. Stiffness, moment of inertia, fracture strength, and Young’s modulus were calculated according to the formulas (equations 1, 2, 3, 4). Fracture stress is defined as the force at the moment of fracture of the bone.

(1)

(2)

(3)

(4)

VOD, vertical outer diameter; HOD, horizontal outer diameter; VID, vertical inner diameter; HID, horizontal inner diameter; P, maximum load (N); L, length between spans; Y, maximum displacement.

Histologic evaluation

For histological analysis, 6 rats were selected from each group and tibia samples were collected. The samples were fixed by soaking in 10% buffered formaldehyde for 2 days, decalcified with decal solution (5% formic acid + 5% hydrochloric acid), and embedded in paraffin after processing in ethanol and xylene. Serial longitudinal 3 μm thick sections were performed with a Slee CUT 6062 microtomes for each sample, and histological standard staining was performed with hematoxylin-eosin (HE) and toluidine blue.

Hematoxylin-eosin stain was used to measure the dimensions of the reactive callus, and toluidine blue stain was used to visualize the fraction of newly formed trabecular bone in the callus and to measure the cartilage fraction in the callus. The level of bone healing was assessed by Allen's grading system [(grades 0 (presence of pseudoarthrosis)-(complete bony union)], which was established for histological assessment of fracture healing.¹⁰ In addition, the grading system defined by Huo et al¹¹ was used in histological evaluation. In this system, 1 point is given for each stage of recovery, 10 points while describing the best recovery and 1 point indicates the most immature. Histological preparations were examined independently by 2 blinded pathologists

Statistical analysis

Statistical Package for Social Sciences 25.0 (IBM SPSS Corp., Armonk, NY, USA) was used for statistical analysis. For pre- and post-experimental weight differences, 2 related samples test (Wilcoxon signed-rank test) was used. For other parameters, mean and standard deviations were calculated with independent sample t-test followed by 2 independent sample test (Mann–Whitney U) to determine statistical differences (P < .05).

Ethical issue

All experimental procedures were performed in the Maltepe University Experimental Animal Application and Research Center. Animal experimental protocol was reviewed and approved by Maltepe University Animal Experiments Ethics Committee (protocol number: 2019.09.01, date: September 16, 2019) and was made in accordance with Turkish Law 6343/2, Veterinary Medicine Deontology Regulation 6.7.26, and with the Helsinki Declaration of Animal Rights.¹²

Results

Surgical intervention was tolerated by all animals. Pain prophylaxis was given to animals 3 days after surgery. There was no wound complication due to individual housing of the rats. In radiologic evaluation, osteolytic zones were observed in some animals. Final x-ray images revealed that callus formation around osteotomy site was satisfactory for all rats. Mean weight of the rats was 492.8 g (standard deviation (SD), 10.5) at the beginning of the study. At the end of the 30th day, mean weight of all rats increased, and it was 502.6 g (SD, 11.2). There was no statistically difference between pre- and post-experimental weight for both groups (control group; P = .084, K2; P = .07).

Radiologic evaluation

Fracture healing process of the groups was evaluated with x-ray weekly and images were analyzed by the same observers. Weekly radiological images of the control group and K2 group were evaluated according to the Lane and Sandhu method. The median score of radiologic fracture healing was 1 (0-1) in the control group and 1 (0-2) in the K2 group in first week which was statistically insignificant (P > .05). However, the median score of radiologic fracture healing was 2 (2-3) in the control group and 3 (3-4) in the K2 group at the last week which reached statistical significance (P = .014) (Table 2) (Figure 3).

Table 2.

Comparison of radiological bone healing by groups

Weeks	Control		K2		P †
	Median	Min.-Max.	Median	Min.-Max.
1st week	1	0-1	1	0-2	.336
2nd week	1	1-2	2	1-2	.093
3rd week	2	2-2	3	2-3	.019*
4th week	2	2-3	3	3-4	.014*

^† Mann–Whitney U, *P <.05.

Min, minimum; max, maximum.

Figure 3.

Radiographic imaging of a rat belonging to the control group at 7 (A), 14 (B), 21 (C), 30 (D) days and K2 group at 7 (E), 14 (F), 21 (G), 30 (H) days.

Biomechanical evaluation

After 30 days, biomechanical test results revealed that K2 group has shown significant increase in parameters. The results of 3-point bending test are shown in Table 3. Breaking force (P = .047), breaking time (P = .019), stiffness (P = .039), fracture strength (P = .041), and Young’s modulus (P = .032) shown statistically significant increase, but breaking displacement difference (P = .063) was not statically significant between control and K2 groups.

Table 3.

Biomechanical test results of control and K2 groups

Parameters	Control		K2		P †
	Median	Min.-Max.	Median	Min.-Max.
Breaking force (stress) (N)	88.1	78.9-96.2	120.1	11.3-133.6	.047*
Breaking displacement (strain) (mm)	2.45	2.2-2.9	2.85	2.5-3.8	.063
Breaking time (seconds)	2.57	2.25-2.9	3.1	2.95-3.45	.019*
Stiffness (N/mm)	119.25	105.4-128.6	158.42	135.8-178.6	.039*
Fracture strength (MPa)	150.01	135.1-163.4	206.88	189.04-274.14	.041*
Young’s modulus (MPa)	7495.1	6706.4-8191.9	10431	9477-25988	.032*

^† Mann–Whitney U, *P <.05.

max, maximum; min, minimum; Mpa, megapascal; N, Newton.

Blood analysis

The results analyzed from blood samples taken during euthanasia are given in Table 4. The analyzed parameters revealed that PICP (P = .024), PINP (P = .047), and osteocalcin (P = .048) levels were significantly higher in K2 group than control group.

Table 4.

Blood analysis of the control and K2 groups

Parameter	Control		K2		P †
	Median	Min.-Max.	Median	Min.-Max.
PICP (ng/mL)	31.79	24.39-50.43	46.49	34.35-52.05	.024*
PINP (pg/mL)	2400	897-3474	3658	1382-4987	.047*
Osteocalcin (ng/mL)	11.46	3.75-13.82	17.17	8.00 -21.88	.048*

^† Mann–Whitney U, *P <.05.

min, minimum; max, maximum; PICP, procollagen I N-terminal propeptid; PINP, procollagen I C-terminal propeptid.

Histologic evaluation

Histologic staining, in which union formation and periosteal callus bridging were examined, revealed that the K2 group had a much more pronounced, denser, and well-organized periosteal callus (Figure 4). However, the scoring system results revealed that there was no statistical difference between the groups. The median value according to Allen's rating score was 2 (min./max.: 1/3) for the control group and 3 (min./max.: 2/4) for the K2 group. There is no statistical difference between the groups (P = .086), Also according to Huo's rating system, the median value was 6.5 (min/max: 5/9) for the control group and 8.5 (7/9) for the K2 group. Again, no static difference was found between the groups (P = .07).

Figure 4.

Callus formation of the control group (A) and K2 group (B).

Discussion

The bone fracture healing process begins with inflammation, which is the initial response at the cellular and vascular level after injury. It continues with the replacement of damaged or dead cells with new cells and repairs of the matrices. It ends with the remodeling process, which involves removing, replacing, and rearranging the repair tissue, often along mechanical stress lines.¹³ Many exogenous and endogenous factors can affect the steps of this process. In recent years, the effects of vitamin K2 on bone metabolism have been better understood by many studies.^14-16 It has an important role in the treatment of osteoporosis and protection of bone structure, especially due to its effects on osteocalcin and bone remodeling.^17-19 The purpose of this preclinic study was to investigate the effect of vitamin K on the bone healing process and the results of the study revealed that MK-7 (vitamin K2 derivative) has been shown to have positive effects on bone healing both biomechanically and histologically.

Animal models are frequently used methods to study the effects of vitamin K2 on bone metabolism. Generally, animal studies about osteoporosis in ovariectomized rats, unilateral sciatic neurectomy rats, or rats with suspended tails found that vitamin K2 has significant positive effects on bone structure maintenance. These studies have shown that vitamin K2 reduces bone mass density and trabecular bone loss, improves osteoblast activity, and increases serum levels of bone anabolic markers.^17,18,20 In addition, a meta-analysis examining the relationship between fracture healing and vitamin K2 by Hao et al²¹ found that vitamin K2 has positive effects on fracture healing. However, the histological and biomechanical bone healing effect of vitamin K2 has not been widely evaluated in the literature.

Increased osteoblastic activity with vitamin K2 treatment strengthens the bone structure. Huang²² reports that γ-carboxylation of osteocalcin can be achieved with vitamin K2 and this carboxylated type of osteocalcin levels can effectively stimulate osteoblast to enhance bone mineralization. According to the bending test results, it was found that the maximum load, breaking load, and elastic load values were increased in rats treated with vitamin K2.²² Matsumoto et al²³ investigated the effect of vitamin K2 on bone material properties and found that cortical bone ultimate strength and toughness increased after vitamin K2 treatment. In our study, a detailed 3-point bending test was applied, and according to the results, a significant difference was found in breaking force, breaking time, stiffness, fracture strength, and Young’s modulus in rats using vitamin K2 compared to the control group.

The positive effects of K2 vitamin on bone metabolism can also be seen when examined at histopathological level. Iwamoto et al²⁴ have stated that vitamin K2 is positively effective in promoting the bone healing process at all stages of fracture healing and remodeling histologically. Another histopathological study on the effects of vitamin K2 on bone metabolism in osteoporotic rats revealed that rats treated with vitamin K2 had larger areas of osteoid, more mineralized bone, and more active osteoblastic activity in the histological sections.²⁵ Our findings about histologic properties of specimens demonstrated that there are no statical differences between groups but bone healing areas of K2 group have more dense and well-organized callus formation than control group. In addition, a statistically significant difference was observed in the radiological follow-ups of the K2 group compared to the control group, starting from the third week.

In studies investigating vitamin K2 therapy, it was observed that with vitamin K2 treatment, carboxylated type of osteocalcin levels increased, osteoblast number increased, and osteoclast number decreased in rats. From this point of view, vitamin K2 has been shown to support bone formation and reduce bone resorption.^26-28 Procollagen I N-terminal propeptid, PICP, and osteocalcin are known as biomarkers of bone formation and fracture healing. Stoffel et al²⁹ found that PICP levels peaked 20-24 weeks after fracture of the tibial shaft. Carter et al³⁰ discovered that PICP levels peaked 2 weeks after distal radial fracture and remained increased at 9 months. Veitch et al³¹ investigated PINP levels and found that PINP levels peaked at 12 weeks after fracture of the tibial shaft remaining increased at 24 weeks. Osteocalcin was main non-collagenous protein produced by osteoblasts and levels were increased at 24 weeks after fracture of the tibial shaft.³² Yuanyang et al³² investigated the effect of vitamin K2 on bone mineral density in female patients with osteoporosis and they found that appropriate vitamin K2 treatment improves bone mineral density in the hip and waist of women with osteoporosis and PINP levels were increased in vitamin K2 treatment in these group. In our study, a remarkable increase was found in biomarkers of bone metabolism such as osteocalcin, PINP, and PICP compared to the control group.

Osteoporotic bone models were mainly studied area for the effects of vitamin K2 on bone metabolism. In our study, since all rats were male, the effects of vitamin K2 on fracture healing could not be evaluated with the hormonal status of female rats and the presence of osteoporosis. In our study, objective radiological comparison, which is usually performed using microcomputed tomography or microfocus radiographs in the literature, could not be made because radiological follow-up was performed by x-ray. Due to the limited duration of the experiment to 4 weeks, limited information has been obtained about the long-term effects of vitamin K2 on callus tissue. These are the main limitations of our study.

In conclusion, it has been revealed that vitamin K2, whose positive effects on the metabolism of osteoporotic bones are well known and clearly demonstrated, positively affects bone metabolism during the fracture union process and creates a more durable callus tissue.