Optimal Intermittent Administration Interval of Parathyroid Hormone 1-34 for Bone Morphogenetic Protein-induced Bone Formation in a Rat Spinal Fusion Model

doi:10.21203/rs.3.rs-54656/v1

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Optimal Intermittent Administration Interval of Parathyroid Hormone 1-34 for Bone Morphogenetic Protein-induced Bone Formation in a Rat Spinal Fusion Model

https://doi.org/10.21203/rs.3.rs-54656/v1

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Background: Both bone morphogenetic protein 2 (BMP-2) and teriparatide (parathyroid hormone [PTH] 1-34) are used to enhance bone healing. There is still no established opinion regarding the optimum dose and administration method. We investigated the optimal dose and administration method for the combination of BMP-2 and PTH 1-34 in a rat spinal fusion model.

Methods: Group I was implanted with a control carrier. Groups II, III, and IV were implanted with a carrier containing 3μg of recombinant human BMP-2 (rhBMP-2). In addition, following implantation, PTH 1-34 injections were administered to Group III thrice a week (total, 180 μg/kg/week) and Group IV six times a week (total, 180 µg/kg/week). The rats were euthanized after 8 weeks, and their spines were explanted; assessed by manual palpation, radiographs, and high-resolution micro-computed tomography (micro-CT); and subjected to histological analysis. Serum markers of bone metabolism were also analyzed.

Results: Manual palpation tests showed that the fusion rates in Groups III and IV were considerably higher than those in Group I. They also had higher radiographic scores than Group I. Micro-CT analysis revealed enhanced bone marrow density expressed as bone volume/tissue volume in Group IV versus Group I. Serum marker analysis revealed that Group IV had higher osteocalcin and lower tartrate-resistant acid phosphatase-5b than Group III. Histological analysis indicated that Group IV had enhanced remodeling.

Conclusions: The rhBMP-2 and PTH combination enhanced bone fusion and remodeling of newly formed bone in the rat spinal fusion model. More frequent administration may be superior in terms of bone fusion and bone metabolism.

Orthopedic Surgery

Parathyroid hormone

bone morphogenetic protein

bone fusion

rat spinal fusion model

There are a few past reports of bone fusion caused by the combination of spinal arthrodesis and various drugs. Spinal arthrodesis is a fundamental treatment option for spinal pathologies and is one of the most common spinal procedures, with more than 20 000 surgeries performed in the United States each year [1]. In this procedure, bone grafts are used to restore mechanical stability to the affected spinal segment by providing bridging bone between vertebrae. Because successful bone fusion between unstable spinal segments leads to pain relief and neurologic recovery, the efficacy of this procedure has gained wide acceptance, and the number of these types of surgery has increased annually with an increase in the aged population [2–5].

Bone morphologic proteins (BMPs) are members of the transforming growth factor-β superfamily [6] and are powerful osteoinductive molecules. BMPs are also considered to promote new osteoclast formation because they stimulate the production of receptor activator of nuclear factor kappa-β ligand osteoblasts and help ensure mature osteoclast survival; therefore, BMPs participate in bone matrix resorption [7–8]. The osteoinductive effects of recombinant human BMP-2 (rhBMP-2) for spinal fusion have been shown in animal models and clinical trials [9–13]. Although BMPs are approved for clinical use, clinical trial results have shown that high doses are required to induce adequate bone fusion because of the following reasons: (1) solubility of the molecules, (2) easy diffusion of the molecules away from the fusion site, and (3) in vivo inactivation [14]. In addition, BMPs are expensive; therefore, their usefulness may be limited by their expense. As a result, a number of strategies are being developed to provide a safer, less expensive, and more efficacious spinal fusion using rhBMP-2.

Teriparatide (parathyroid hormone [PTH) 1–34] is an anabolic drug, and its efficacy as an osteoporosis drug has been widely verified through experimental and clinical studies [15–18]. Although PTH can have bone resorption effects with continuous administration, it can also accelerate bone formation with intermittent administration. PTH 1–34 could also enhance fracture healing not only in ovariectomized rats, but also in intact rats [19, 20]. The clinical use of PTH 1–34 to accelerate fracture healing has been attempted, and the combination of PTH 1–34 and BMPs has been shown to favor spinal fusion [21]. However, the optimal intermittent administration interval of PTH1-34 has not been revealed.

We previously reported that the synergistic effect of rhBMP-2 and PTH 1–34 administered systemically as a single dose at the optimal time was efficacious for fracture repair and significantly enhanced bone fusion in a rat femoral fracture model [22]. The purpose of this study was to clarify the optimal dosing interval and administration method for the combination of rhBMP-2 and PTH 1–34 in a rat spinal fusion model.

Preparation of Matrices

CollaCote® (Zimmer/dental) is a biodegradable collagen scaffolding used for cellular attachment. Although a collagen sponge has been used clinically as a carrier for rhBMP-2 [23, 24], CollaCote® has an allograft matrix possessing hemostatic function that facilitates early clot formation and wound stabilization. The final concentration of rhBMP-2 (Peprotech, Rocky Hill, NJ) was dissolved in phosphate buffered saline (pH 7.5) and applied to CollaCote®. CollaCote® was cut with a scalpel into 5 mm × 20 mm strips and placed with rhBMP-2 in an Eppendorf tube that was left overnight at 4 °C prior to implantation. Similarly, 100 µL of rhBMP-2-free phosphate buffered saline was added to CollaCote® to obtain rhBMP-2-free CollaCote®.

Animals

All animal studies were approved by the Oita University Animal Research Committee, and experiments conformed to all guidelines and regulations for the protection of animal welfare (protocol No. 1624002).

Study Groups

A total of 52 male Sprague–Dawley rats (8–10 weeks old; CLEA Japan, Inc., Tokyo, Japan) were divided into four groups. Group I (n = 10) included animals that were implanted with a control carrier alone. Group II (n = 14) included animals that were implanted with carriers containing 3 µg of rhBMP-2. Group III (n = 14) included animals that were implanted with carriers containing 3 µg of rhBMP-2, followed by injections of PTH 1–34 (Teribone; Asahi Kasei Pharma, Tokyo, Japan) (60 µg/kg) three times a week (total, 180 µg//kg /week). Group IV (n = 14) included animals that were implanted with a carrier containing 3 µg of rhBMP-2, followed by injections of PTH 1–34 (30 µg/kg) six times a week (total, 180 µg/kg/week). Rats in groups III and IV were subcutaneously injected with PTH beginning 1 week after surgery. The injections were continued until immediately before the rats were euthanized.

Surgical Technique for Constructing the L4–L5 Posterolateral Spinal Fusion Model

A posterior midline incision was made on the skin. Next, two separate paramedian incisions were made 3 mm from the midline in the lumbar fascia, and the transverse processes were exposed. The transverse processes of L4 and L5 were decorticated using a low-speed burr. Subsequently, CollaCote® with or without rhBMP-2 was implanted on each side. The fascial and skin incisions were closed with a 3–0 absorbable suture. Immediately following surgery and on subsequent days, the rodents received analgesics (buprenorphine subcutaneously and paracetamol). The rodents were housed in separate cages and fed food and water ad libitum, and their condition was monitored on a daily basis. The rats were humanely euthanized 8 weeks postoperatively.

Manual Assessment of Fusion

Eight weeks post-implantation, the explanted spines were manually tested for intersegmental motion by three blinded independent observers [25–31]. The explanted lumbar spine was palpated gently, and the lateral side bending motion at the L4–L5 level was compared with the motion at the adjacent levels above (L3–L4) and below (L5–L6). The absence of motion was considered a successful fusion. Any motion detected between the transverse processes was considered a failure of fusion. The spine was designated as “not fused” if any of the three observers graded the spine as not fused. The spines were scored as either fused on both the right and left sides or not fused, and then, the fusion rate was calculated.

Radiographic Analysis

The explanted spines obtained at the 8-week time point were photographed using a Softex X-ray apparatus (Softex CSM-2; Softex, Tokyo, Japan) employing HS Fuji Softex film (Fuji Film, Tokyo, Japan) at 45 cm with 30 kV and 15 mA for 20 s. The fusion between the L4 and L5 transverse processes in each rat was recorded as a percentage of the total area between the L4 and L5 that was filled with new bone [25]. Three blinded independent observers scored the bone formation in each rat using a 5-point scale: 0 = no bone formation; 1 = bone filling in less than 25% of the area; 2 = bone filling in 25–50% of the area; 3 = bone filling in 50–75% of the area; and 4 = bone filling in 75–100% of the area. The spines were scored on both the right and left sides.

Micro-CT Analysis

The spines were scanned by micro-CT using SkyScan 1172 (Bruker microCT, Kontich, Belgium) with a voxel size of 20 mm. The data were collected at 100 kV and 100 mA and reconstructed using the cone-beam algorithm. Each spine was set on the object stage, and sample scanning was performed over a 180° rotation with an exposure time of 105 ms. A cylindrical volume of interest with a diameter of 20 mm and a height of 27 mm, which displayed the micro-structure of the rat vertebra as comprising cortical and cancellous bone, was selected. Data analysis of the area from the top of the L4 transverse processes to the bottom of the L5 transverse processes, including the vertebrae, was performed using CT Analyzer software (Bruker microCT). The spines were analyzed on both the right and left sides. Bone mineral density, tissue volume, bone volume, trabecular thickness, trabecular spacing, and bone volume fraction (bone volume/tissue volume, %) were measured.

Analysis of Serum Markers of Bone Metabolism

Just prior to euthanization of the animals, blood samples were collected and stored at -80 °C until analysis. Serum markers of bone metabolism were analyzed using an enzyme-linked immunosorbent assay specific for osteocalcin (Osteocalcin High Sensitive EIA Kit [rat]; Takara Bio, Shiga, Japan) and tartrate-resistant acid phosphatase-5b (TRACP5b) (RatTRAP Assay; Immunodiagnostic Systems Ltd., Boldon, UK) according to the manufacturer’s instructions.

Histological Analysis

Eight weeks after implantation, the spines were dissected and the specimens were fixed in 40% ethanol, decalcified using standard 10% decalcifying solution HCI (Cal-Ex; Fischer Scientific, Fairlawn, NJ), washed with running tap water, and then transferred to 75% ethanol. Serial sagittal sections near the transverse processes were carefully cut at the level of the transverse process on both the right and left sides. The specimens were embedded in wax for sectioning. Sagittal sections (5 mm) were cut from the paraffin blocks using a microtome (LS-113; DAIWA-KOKI, Saitama, Japan). The sections were stained with hematoxylin and eosin for basic morphology. Three blinded independent observers scored histological bone formation. Histological fusion was defined as bony trabeculae bridging from one transverse process to the next.²⁵ Fusion masses were assessed, and the extent of new bone formation was scored using the following scoring criteria: 1 = fibrocartilage tissue filling less than 25% of the gap area; 2 = fibrocartilage tissue filling 25–75% of the gap area; 3 = fibrocartilage and bone tissue filling 75–99% of the gap area; 4 = bridged with bone tissue, but the fusion masses comprise thin trabecular bone; and 5 = completely bridged with abundant mature bone tissue. The spines were scored on both the right and left sides.

Statistical Methods

The Statistical Package for the Social Sciences (SPSS) (V13; IBM Corporation, Armonk, NY) was used for statistical analysis. Analysis of variance was used for statistical analysis. P-values < 0.05 were considered significant. The kappa statistic was calculated as a measure of the interobserver reliability of the three independent blinded observers. The kappa statistic corrects the observed agreement for possible chance agreement among observers. Agreement was rated as follows: poor, κ = 0–0.20; fair, κ = 0.21–0.40; moderate, κ = 0.41–0.60; substantial, κ = 0.61–0.80; and excellent, κ > 0.81. A value of 1 indicated absolute agreement, whereas a value of 0 indicated agreement no better than chance.

No abnormal behavior or neurological deficits were noted in any of the 52 rats before or after the surgical procedure or at the time of euthanasia.

Manual Palpation

Table 1 shows the proportion of subjects in each group who achieved fusion according to the three independent evaluators. Consistent agreement (κ = 0.864) was noted among the three independent observers who performed manual palpation.

Eight segments in Group III (n = 14, segments = 28) were assessed as fused (fusion rate, 28.6%) and 10 segments in Group IV (n = 14, segments = 28) exhibited fusion (fusion rate, 35.7%), whereas two segments in Group II (n = 14, segments = 28) exhibited fusion (fusion rate, 7.1%). None of the spines in Group I (n = 10, segments = 20) were fused (fusion rate, 0%). The subjects in Groups III and IV had a higher fusion rates than those in Groups I and II. There was no signiﬁcant difference between the manual assessment scores of Groups I and II, whereas signiﬁcantly higher fusion rates were observed in Groups III and IV than in Group I (p < 0.05).

Radiographic Analysis

Radiographs of the spines were obtained at 8 weeks. Consistent agreement (κ = 0.812) was noted among the three independent observers who graded the radiographs. The spines were scored on both the right and left sides. The average evaluation scores for each group are shown in Table 2, and the representative anteroposterior radiographs of the representative case in each group at 8 weeks are shown in Figure 1. At 8 weeks postoperatively, Group III and IV showed evidence of bone formation between the L4 and L5 transverse processes, and bony bridging was detected. Mineralized callus bridging between the L4 and L5 transverse processes was detected in Group II, although the amount of callus was deemed insufﬁcient. Group I showed no evidence of bone formation. The scores of Groups III and IV were signiﬁcantly higher than those of Group I (p < 0.05).

Micro-CT Analysis

A computer analysis of the micro-CT images revealed the volume of new bone and the quality of the spinal fusion area. The average micro-CT data based on the histomorphometry of each group are shown in Table 3. Analysis revealed a statistical difference in the variance in the bone volume percentage. The bone volume percentage of Group IV was larger than that of Group I (p = 0.023). Trabecular thickness (p = 0.041) and trabecular spacing (p = 0.026) were also significantly different between Groups I and IV. Trabecular thickness (p = 0.040) was statistically significant between Groups I and III. There were no differences between each group regarding tissue volume or bone volume.

Serum Markers of Bone Metabolism

Enzyme-linked immunosorbent assay demonstrated that serum levels of osteocalcin did not differ significantly between Groups II, III, and IV compared with those in Group I. In contrast, a significant difference was found between Group III and Group IV (p < 0.05) (Figure 2). Similarly, serum levels of TRACP5b did not differ significantly between Groups II, III, and IV compared with those in Group I. However, a significant difference was found between Group III and Group IV (p < 0.05) (Figure 3).

Histological Analysis

Histological analysis of Group I showed a paucity of new bone formation and no evidence of fusion (Figure 4A, B). These images clearly demonstrate the muscle between the transverse processes for both specimens. Occasional evidence of new bone formation was observed, originating either from the decorticated transverse process or normal remodeling. A representative case in Group II showed distribution of cartilaginous tissue; however, there was ﬁbrosis tissue and muscle ﬁber between the transverse processes and no evidence of bone fusion (Figure 5A, B). Representative cases in Group III showed distribution of cartilaginous tissue and immature bone formation, but no woven bone between the transverse processes (Figure 6A, B). A representative case in Group IV showed new bone formation bridging the transverse processes, demonstrating mature osteoid tissue and contracting trabeculae (Figure 7A, B). Histological fusion scores are shown in Table 5. The spines were scored on both the right and left sides. Groups III and IV had signiﬁcantly higher histological scores than Group I (p < 0.05). Consistent agreement (κ = 0.862) was noted among the three independent observers.

In the present study, we investigated the optimal intermittent administration interval dosing of PTH 1–34 using rhBMPs for spinal fusion in a rat spinal fusion model. Posterolateral lumbar fusion in rats has been well established as an acceptable model for measuring bone growth, and manual palpation is the most sensitive and specific method for assessing spinal fusion [25–32]. Our previous study reported that 3 µg of rhBMP-2 was insufficient to achieve 100% bone fusion in this model [25] Therefore, 3 µg of BMP was appropriate for testing the optimal intermittent administration interval dosing of PTH 1–34. Although a generous amount of PTH 1–34 effectively demonstrated fracture healing in rat models [19], the clinical dose for humans must be much higher. The clinical dose of PTH 1–34 to treat human osteoporosis is 20–40 µg daily in Japan and the USA. A dose of 5 µg/kg/day seemed ineffective for changing the mechanical properties in a rat [20]. Morimoto et al. [33] reported successful spinal fusion with weekly doses of 180 µg/kg/week, PTH 1–34 in a rat model of spinal fusion. Therefore, we adopted 180 µg/kg/week (3 times/week and 6 times/week). Owing to the difference in metabolism between humans and rats, it is assumed that the 6 times weekly administration in rats is approximately 2 times weekly administration in humans, and the 3 times weekly administration in rats is approximately once weekly administration in humans [34].

Recent studies of the interactions between PTH and BMP showed two types of interactions between PTH and rhBMP-2 [35]. The first is a mechanism that occurs through a complex comprising PTH type-1 receptors, low-density lipoprotein receptor-related protein 6 within the canonical Wnt pathway, and BMP antagonists. Endocytosis of the complex suppresses negative feedback of BMP antagonists, thereby stimulating BMP signaling. Thus, PTH enhances the differentiation of mesenchymal stem cells into osteoblasts. The second is a mechanism that occurs through peroxisome proliferator-activated receptor gamma (PPARγ). Excessive BMP signaling results in downregulation of the Wnt pathway through sclerostin. The downregulated Wnt pathway results in increased PPARγ, which persuades mesenchymal stem cells to differentiate into adipocytes. PTH can activate the Wnt pathway and suppress PPARγ. Morimoto et al. [33] reported synergistic anabolic effects with the concomitant use of intermittent PTH 1–34 and locally applied rhBMP-2 in a rat model of spinal fusion. In their study, the combination increased fusion rates and improved the quality of the newly formed bone. In the present study, Groups III and IV had significantly better fusion rates than Group I according to the manual palpation tests and higher radiographic evaluation scores. They also had a better fusion rate than Group II. Micro-CT analysis indicated that Groups III and IV had significantly higher bone volume/tissue volume scores than those of Group I, which indicated increased bone marrow density. The trabecular thickness and trabecular number also showed statistical differences. Therefore, PTH 1–34 has the effect of strengthening the trabecular bone structure, and the effect may be stronger when administered 6 times a week. Although no significant difference was observed, Group IV tended to be superior to Group III.

Earlier studies investigating the long-term daily administration of PTH 1–34 in ovariectomized rats [36, 37] did not report its effects on bone metabolism markers. In a previous study, daily administration of PTH 1–34 at a dose of 30 µg/kg for 12 months led to a 40% increase in osteocalcin, a bone formation marker, relative to that in the control group [38]. In our study, six-times-weekly administration of PTH 1–34 at 30 µg/kg (180 µg/kg/week) elicited a similar increase in osteocalcin. We observed a decrease in serum TRACP5b following six-times-weekly administration of PTH 1–34 compared with that in the control group. In contrast, in the three-times-weekly administration group, neither osteocalcin nor TRACP5b was significantly different from that in the control group. These results suggest that six-times-weekly administration of PTH 1–34 may enhance bone formation with no accompanying increase in bone resorption. Therefore, six times a week dosing may be the best based on the results of bone metabolism markers in the present study.

The results of the histological analysis revealed that Groups III and IV had well-developed bone trabeculae and bone marrow and mature osteocytes that were not seen in Groups I and II. Group IV also showed an almost normal appearance of the bone marrow compared to Group III. This could be evidence of enhanced remodeling caused by more frequent PTH 1–34 administration.

The limitations of the present study were that we could not see the bone fusion process because we decided to sacrifice the rats at 8 weeks after surgery, when complete bone fusion of the spine had occurred. Despite this, we chose to use a practical dose of rhBMP-2 and PTH 1–34 in the clinical setting, which was advantageous to the study. Although we cannot directly apply our findings to humans because the rat has different biological reactions to drugs, we believe that the results of the current study provide further evidence for understanding the effects of these therapeutic agents.

In conclusion, the combination of rhBMP-2 and PTH 1–34 enhanced bone fusion and remodeling of newly formed bone in a rat spinal fusion model. More frequent administration may be superior in terms of bone formation.

BMP-2

Bone morphogenetic protein 2; BMPs:Bone morphogenetic proteins; BV:bone volume; PBS:phosphate-buffered saline; PPARγ:peroxisome proliferator-activated receptor gamma; PTH:Parathyroid hormone; rhBMPs:recombinant human BMPs; Tb.N:trabecular number; Tb.Th:Trabecular thickness; TV:tissue volume

Acknowledgements

We are grateful to Miss Fumiko Shiba for excellent technical assistance.

Funding

This work was supported by JSPS KAKENHI Grant Number JP16K10829.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors’ contributions

TA, SK, and TI carried out the operation. HT conceived of the study design. SK, MM, YT, MK, and HT interpreted the data and drafted the manuscript. All authors read and approved the final manuscript.

Ethics approval

This study was conducted in accordance with the Declaration of Helsinki. Approval was obtained from the Oita University animal research committee prior to animal experimentation (Oita University institutional Animal Ethics Committee no.1624002).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Table 1. Assessment of Spinal Fusion Via Manual Palpation.

Treatment Group		No. Assessed Manually for Fusion	No. Assessed as Fused	Fusion Rate (%)
Group Ⅰ	Carrier alone	20	0	0
Group Ⅱ	3μg rhBMP-2	28	2	7.1
Group Ⅲ	3μg rhBMP-2 + PTH　(3 times/week)	28	8	28.6*
Group Ⅳ	3μg rhBMP-2 + PTH　(6 times/week)	28	10	35.7*

*p < 0.05 (vs Group I)

Table 2. Radiographic Scores at 8 weeks.

Treatment Group		No. studied radiographically	Score at 8 weeks (mean ± SD)
Group Ⅰ	Carrier alone	20	0.11 ± 1.41
Group Ⅱ	3μg rhBMP-2	28	0.48 ± 1.55
Group Ⅲ	3μg rhBMP-2 + PTH (3 times/week)	28	1.75 ± 1.37*
Group Ⅳ	3μg rhBMP-2 + PTH (6 times/week)	28	2.11 ± 1.21*

*p < 0.05 (vs Group I)

Table 3.1. Micro-CT Based Histomorphometry of Spines at 8 weeks.

Treatment Group		TV (mm³)	BV (mm³)	BV/TV (%)
Group Ⅰ	Carrier alone	123.71 ± 6.88	47.44 ± 8.56	38.56 ± 7.78
Group Ⅱ	3μg rhBMP-2	119.87 ± 13.30	44.19 ± 9.45	37.05 ± 7.77
Group Ⅲ	3μg rhBMP-2 + PTH (3 times/week)	114.96 ± 7.45	49.19 ± 8.80	42.97 ± 8.29
Group Ⅳ	3μg rhBMP-2 + PTH (6 times/week)	108.13 ± 12.84	58.82 ± 9.68	54.20 ± 2.49*

TV, tissue volume; BV, bone volume; BV/TV, bone volume fraction.

*p < 0.05 (vs Group I)

Table 3.2. Micro-CT based histomorphometry of spines at 8 weeks.

Treatment Group		Tb. Th (mm)	Tb. N (1/mm)	Tb. Sp (1/mm)
Group Ⅰ	Carrier alone	0.22 ± 0.05	1.70 ± 0.04	0.41 ± 0.11
Group Ⅱ	3μg rhBMP-2	0.21 ± 0.06	1.81 ± 0.14	0.46 ± 0.13
Group Ⅲ	3μg rhBMP-2 + PTH (3 times/week)	0.21 ± 0.06	2.11 ± 0.31*	0.38 ± 0.14
Group Ⅳ	3μg rhBMP-2 + PTH (6 times/week)	0.30 ± 0.03*	1.84 ± 0.29	0.20 ± 0.05*

Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular spacing.

*p < 0.05 (vs Group I)

Table 4. Histologic fusion score at 8 weeks.

Treatment Group		Score at 8 weeks (mean ± SD)
Group Ⅰ	Carrier alone	0.10 ± 1.02
Group Ⅱ	3μg rhBMP-2	0.68 ± 1.33
Group Ⅲ	3μg rhBMP-2 + PTH (3 times/week)	2.66 ± 1.21*
Group Ⅳ	3μg rhBMP-2 + PTH (6 times/week)	3.15 ± 1.73*

*p < 0.05 (vs Group I)

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Editorial decision: Minor revision
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Optimal Intermittent Administration Interval of Parathyroid Hormone 1-34 for Bone Morphogenetic Protein-induced Bone Formation in a Rat Spinal Fusion Model

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Version 1

Abstract

Figures

Background

Methods

Preparation of Matrices

Animals

Study Groups

Surgical Technique for Constructing the L4–L5 Posterolateral Spinal Fusion Model

Manual Assessment of Fusion

Radiographic Analysis

Micro-CT Analysis

Analysis of Serum Markers of Bone Metabolism

Histological Analysis

Statistical Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Tables

Status:

Version 1