Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique

Ding Li; Dun Zhao; Zhikui Zeng; Feng Huang; Ziwei Jiang; Hao Xiong; Tianan Guan; Bin Fang; Yue Li

doi:10.1371/journal.pone.0278688

. 2022 Dec 6;17(12):e0278688. doi: 10.1371/journal.pone.0278688

Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique

Ding Li ¹, Dun Zhao ¹, Zhikui Zeng ², Feng Huang ¹, Ziwei Jiang ¹, Hao Xiong ¹, Tianan Guan ¹, Bin Fang ¹, Yue Li ^1,^*

Editor: Selvaraj Vimalraj³

PMCID: PMC9725127 PMID: 36473008

Abstract

Context

Rhizoma drynariae total flavonoids (RDTF) are used to treat fractures. CD31^hiEmcn^hi vessels induced by PDGF-BB secreted by osteoclast precursors, together with osteoblasts and osteoclasts, constitute the ternary regulatory mechanism of bone tissue reconstruction.

Objective

This study aimed to determine whether RDTF can promote bone tissue remodeling and induce membrane growth in the rat Masquelet model and to explore its molecular mechanism based on the ternary regulation theory.

Methods

Thirty-six SD rats were randomized to three groups: blank, induced membrane, and RDTF treatment (n = 12/group). The gross morphological characteristics of the new bone tissue were observed after 6 weeks. Sixty SD rats were also randomized to five groups: blank, induction membrane, low-dose RDTF, medium-dose RDTF, and high-dose RDTF (n = 12/group). After 4 weeks, immunohistochemistry and western blot were used to detect the expression of membrane tissue-related proteins. The mRNA expression of key factors of ternary regulation was analyzed by qRT-PCR.

Results

RDTF positively affected angiogenesis and bone tissue reconstruction in the bone defect area. RDTF could upregulate the expression of key factors (PDGF-BB, CD31, and endomucin), VEGF, and HMGB1 mRNA and proteins in the ternary regulation pathway.

Discussion and conclusion

Although the expected CD31^hiEmcn^hi vessels in the induction membrane were not observed, this study confirmed that RDTF could promote the secretion of angiogenic factors in the induced membrane. The specific mechanisms still need to be further studied.

1 Introduction

Bone defects caused by severe trauma, infection, tumor resection, and other reasons are common in clinical practice, which seriously affects patients’ quality of life and causes significant economic and social burden. The treatment of bone defect is a challenge for the surgeons, numerous options are use in the clinical such as free cancellous bone grafts, allogeneic bone, vascularized fibula graft and distraction osteogenesis. But these techniques have their limitations, like bone resorption, long period of treatment, delayed union or nonunion. The free cancellous bone grafts and vascularized fibula graft are often used to repair small size bone defects, but not for critical bone defects (defects >6 cm), [1]. Allogeneic bone and artificial bone are highly dependent upon vascularization, use the traditional surgery technique, it is difficult to achieve a sufficient blood supply [2, 3]. Distraction osteogenesis is an efficient technique to treat the critical bone defects, but the long period of treatment made the patients suffering a painful process.[4–7]. How to improve the efficiency and shorten the period of treatment is a challenge for the orthopedist.

In 1986, Masquelet proposed the concept of “induced membrane” to prevent the invasion of adjacent soft tissues into the bone defect, maintain the autograft in place, preserve the autograft volume and producing osteoinductive substances locally [8–10]. The Masquelet technique is a two-stage procedure, the first stage involved debridement of bone and soft tissues, insertion of a polymethylmethacrylate (PMMA) cement spacer, bone stabilization.After 4 to 8 weeks, a membrane surrounding the spacer was formed and then the second stage surgery was operated to remove the spacer for implantation of bone grafts. A series of reports have showed that Masquelet technique can be used on the reconstruction of bone defect. However, it also has disadvantages such as poor osteogenesis quality of new bone, and infection [10–12], joint contracture, bone nonunion, and stress fracture [13]. The formation of the induced membrane and its ability to induce bone remodeling is the key point of the Masquelet technique. The mesenchymal stem cells (MSCs) from the induced membrane and normal bone marrow show the same osteogenic potential [14]. The membrane are rich in osteoblasts, osteoclasts, osteoclast precursor cells, secrete vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β1), bone morphogenetic protein-2 (BMP-2), and other growth factors that promote bone formation [10, 15]. CD31^hiEmcn^hi vessels play an important role in bone reconstruction [16]. During the process of bone formation and resorption, the decrease of CD31^hiEmcn^hi angiogenesis results in a significant reduction in the number of trabecular and cortical bones [16]. CD31^hiEmcn^hi angiogenesis is primarily regulated by platelet-derived growth factor-BB (PDGF-BB). PDGF-BB can induce CD31^hiEmcn^hi angiogenesis by promoting endothelial progenitor cells (EPCs) and MSCs. For this reason, CD31^hiEmcn^hi vessels, along with osteoclasts and osteoblasts, constitute the “ternary regulation” mechanism of bone remodeling.

Rhizoma drynariae (Chinese name, Gusuibu) is a traditional Chinese herb with a variety of pharmacological activities [17, 18] and is used for the prevention or treatment of bone-related diseases [19, 20]. The major bioactive constituents of are flavonoids, with potential roles in osteoporosis prevention and the treatment of disc degeneration [21, 22]. Rhizoma drynariae total flavonoids (RDTF) have some efficacy for improving proliferation and differentiation [23, 24], inflammation [25], fracture healing, and teeth osteocyte protection [26]. But, whether the RDTF can improve the efficiency of Masquelet technique is still unknown.

In this study we focused on whether RDTF can promote induce membrane growth and bone remodeling in the rat Masquelet model and explore its molecular mechanism based on ternary regulation theory.

2 Material and methods

2.1 Animals and Masquelet’s technique model establishment

Masquelet’s technique was performed in a rat femur defect model. Totally ninety-six specific pathogen-free (SPF) male rats (8–10 weeks of age, weighing 260–280 g) were provided by the Laboratory Animal Center of Guangzhou University of Traditional Chinese Medicine(ethical number: SCX-2018-0034). All animals were treated in compliance with the principles and procedures contained in the most recent publication of the National Institutes of Health guide for the care and use of laboratory animals. The rats were housed four animals/cage, at 24–26°C and 60–80% humidity, with 14 h of light/day. They received rat chow and water ad libitum. For the stage one, rats were general intraperitoneal anesthesia (5% chloral hydrate, 8 mg/kg), the right hindlimb was shaved, and the animals were placed in a lateral position. Penicillin (80,000 U, North China Pharmaceutical Co. Ltd, China) was injected intramuscularly before surgery to prevention of infection. A longitudinal incision was made in the skin and superficial fascia over the right femur. The biceps femoris and vastus lateralis muscles were separated and elevated from the greater trochanter, exposing the lateral aspect of the femoral bone. A six-hole, 1.5-mm homemade stainless steel mini-plate was applied to the lateral aspect of the femur shaft. Two 1.5-mm cortical screws (Suzhou Yuelai Medical Equipment Co., Ltd., Jiangsu, China) were drilled in holes #1 and #6 for fixation (S1 Fig). Then, two Kirshner wires were drilled into holes #2 and #5. The osteotomy guide plate was placed above the steel plate after passing through the holes. With the osteotomy guide plate was secured, a critical-sized defect (CSD) measuring 4 mm was created in the femur bone shaft, using a reciprocating saw. The bone defect was filled with PMMA cement and molded into a cylindrical shape. After securing the cement with silk thread, the wound was washed with sterile water, and the fascia and skin were sewn. 4 weeks after the stage one surgery the stage 2 surgery was conducted, preoperative preparation and surgical approach were the same as that of the first stage. The induced membrane was carefully cut open, PMMA cement was removed, and two segments of rat coccyx were taken as bone graft materials to implant in the bone defect area. The induced membrane tissue was sutured, and muscle, deep fascia, and skin were sutured in turn. The animals were returned to their cages and house separated. Penicillin was given for 5 days postoperatively, during which time the rats were monitored daily for the occurrence of abnormal behavior and/or complications. Meloxicam (0.2mg/kg) in the first 3 days after operative to analgesia. Under general anesthesia, the membranes surrounding PMMA cement spacer were harvested 4 weeks after the first stage. After the experiment, the animals were euthanized with an overdose (150 mg/kg) of pentobarbital administered intraperitoneally.

2.2 Treatment

Experiment 1, 60 rats were randomly divided into five groups (empty control group, EG; induced membrane group, IG; low dose RDTF group, LG; medium dose group, MG; high dose group, HG). The operation for the bone defect was performed as above, and no second stage surgery was performed. The EG group was not implanted with PMMA cement. Approximately 24 h after the animal model was established, the LG, MG, and HG groups were administered with QiangGu capsules (contain 180mg RDTF/each, National Medicine Permit No. Z20030007; batch #180304) intragastrically. Mixed the powder in distilled water, and administered by gavage, the intervention concentrations were 157.5 mg/kg/d for HG, 78.75 mg/kg/d for MG, and 39.38 mg/kg/d for LG. The animals in the EG and IG groups were given the same amount of pure water. Gavage was started on the first day after modeling and was performed once a day until the sampling day of the fourth week after surgery. Histological and radiographs were performed routinely at 4 weeks postoperatively. The rats with complications were not included in the analyses to avoid confounding factors from infections and inflammation.

Experiment 2, 36 rats were randomly divided into three groups (empty control group, ECG; induced membrane group, IMG; induced membrane plus total flavonoids group, IFG). The operation process of the bone defect was performed as above. The ECG group was not implanted with PMMA cement. The second stage surgery was performed 4 weeks after PMMA cement implantation. Bone grafting was not performed in the ECG group, and the postoperative treatment was the same as that in the first stage. QiangGu capsules (contain 180mg RDTF/each, National Medicine Permit No. Z20030007; batch #180304) were used. Approximately 24 hours after the animal model was established, the IMG and IFG groups were administered QiangGu capsules mixed in distilled water., the dose was 78.75 mg/kg/d, The ECG and IMG groups were fed the same dose of 0.9% normal saline intragastrically every day for six weeks. All 36 rats tolerated the operation and were kept in single cages during the experiment.

2.3 Micro CT and angiography analyses

After general anesthesia with an intraperitoneal injection of 5% (w/v) chloral hydrate (0.8 ml/100 g), anteroposterior and lateral radiographs of the right hindlimb were obtained using X-ray (50 kV and 4 mA; Kodak, Rochester, NY, USA) at 4 weeks after the operation. For the ECG, IMG, and IFG, the radiographs were obtained 6 weeks after the second-stage surgery. The radiographs were scored using the Lane-Sandhu scoring system. Scoring was carried out by three experts in the department of radiology.

For angiography, after anesthesia, the skin and subcutaneous tissues were incised under the xiphoid process of the sternum. The heart was exposed by an incision along the manubrium sternum and ribs. The perfusion needle was inserted from the right atrium to reach the aortic arch. The left atrial appendage was cut open, and 1% heparin saline was perfused until the fluid was clear and there was no blood outflow. After paraldehyde infusion and fixation, the Microfil (Microfil, MV-122, Flow Tech, Inc.) contrast agent was injected until it was completely drained (nearly 20 ml), put the animal in 4°C overnight, and then collected the femur, decalcification thoroughly. Use the Micro-CT to scan and analysis.

For the Micro-CT scan, femur were collected, and the attached soft tissue was removed thoroughly and then fixed in 4% paraformaldehyde. The specimens were scanned with an animal micro-CT scanner (Skyscan 1172, Bruker, Madison, WI, USA). The Scanning parameters with an 80 kVp energy setting, intensity of 313 uA with 280 ms acquisition time and a image resolution of 120 μm. The defective region was identified by a contour as a traced region of interest (ROI).

2.4 Histoological analysis

Histological evaluations of the induced membranes were performed by microscope observations of stained tissue sections. Cryosections (5-μm thick) of the induced membrane were stained with hematoxylin and eosin (H&E) (Solaibao Technology Co., Ltd., Beijing, China). The components of the extracellular membrane were detected with an Elastica-van Gieson stain (Merck Serono KGaA, Darmstadt, Germany) and Muscovy O-solid green cartilage staining (Feijing Biotechnology Co., Ltd., Beijing, China), according to the manufacturers’ instructions.

2.5 Quantitative real-time polymerase chain reaction (qRT-PCR)

Pieces of membranes were immediately frozen after collection and stored in liquid nitrogen. Frozen membrane (200–600 mg) was powdered with a pestle and homogenized in TRIzol (Promega, Madison, WI, USA). RNA was used for cDNA synthesis with the RevertAid First-Strand Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) using oligo-dT as a primer. cDNA product (1 μl) was used for qRT-PCR with the SYBER Green technique (SYBR Green/ROX qPCR Master Mix) by quantifying the product at each cycle in a real-time manner. The amount of product was monitored for 40 cycles. The product specificity was confirmed by melting curve and, in some cases, by visualizing the products with agarose gel electrophoresis. The expression of β-actin was studied using the primer 5’-GAT CAA GAT CAT TGC TCC TCC TG-3’ and 5’-AGG GTG TAA AAC GCA GCT CA-3’ (annealing temperature of 60°C). The primers were 5’-CAC CCA AGT GTT TTG GCA CC-3’ and 5’-ATG AGC CCT TTC TTC CAC GG-3’ for CD31, 5’-GTG CAA CAC CCA AAA CAC CT-3’ and 5’-TGA ACT CTG ATT CTC CGT CTT GT-3’ for Endomucin, 5’-GTC GGA GAG CAA CGT CAC TA-3’ and 5’-TGC GCT TTC GTT TTT GAC CC-3’ for VEGF, 5’-ACT TGA ACA TGA CCC GAG CA-3’ and 5’-GAA CAC CTC TGT ACG CGT CT-3’ for PDGF-BB, and 5’-ACC GCC CAA AAA TCA AAG GC-3’ and 5’-ATA GGG CTG CTT GTC ATC CG-3’ for HMGB1, all with an annealing temperature of 66°C. GADPH was used as endogenous controls, relative expression level was computed using 2-ΔΔCt method.

2.6 Western-blot

The induced membrane proteins were collected by lysis in RIPA buffer, a BCA protein assay was used to measure the protein content. After centrifugation for 10 min, the extracted protein was put in boiling water for 10 min. The proteins were isolated by 10% polyacrylamide gel electrophoresis at 120 mV, and transferred to PVDF membranes at 200 mA for approximately 1h. Subsequently, the membranes were blocked using 5% non-fat dried milk and 4% BSA for 2 h at 25°C, followed by incubation with the following primary antibodies: anti-GAPDH (1:1000, Shanghai Kangcheng Co., China), anti- HMGB1 (1:1000, ab79823, Abcam, Cambridge, United Kingdom), anti-VEGFA (1:1000, ab46154, Abcam, Cambridge, United Kingdom), anti-CD31 (1:1000, ab182981, Abcam, Cambridge, United Kingdom), and anti-PDGF-BB (1:1000, ab16829, Abcam, Cambridge, United Kingdom), anti-Endomucin (1:1000, sc-65495, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000, ZB-2301, Wuhan Bode Biological Engineering Co, Ltd, Wuhan, China) for 1h on a shaker at 37°C. Finally, after ECL chemiluminescence, development, and fixation, the film was scanned. Sensiansys software in a JS-680A automatic gel imaging analysis system was used for the grayscale analysis of the target bands.

2.7 Immunohistochemistry

Sections were obtained as mentioned above, and the slides were incubated overnight. The slides were washed for 15 min in 0.1% Triton X-100 diluted with PBS and blocked with 5% bovine serum albumin (ZSGB-BIO, Beijing, China) for 30 min. The slides were incubated in the following antibodies overnight at 4°C: CD31 anti-collagen I (1:2000, ab182981, Abcam, Cambridge, United Kingdom), endomucin anti-collagen I (1:50, sc-65495, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PDGF-BB anti-collagen I (1:100, ab16829, Abcam, Cambridge, United Kingdom), VEGFA anti-collagen I (1:100, ab46154, Abcam, Cambridge, United Kingdom), HMGB1 anti-collagen I (1:100, ab79823, Abcam, Cambridge, United Kingdom). The slides were washed with PBS AND incubated with a fluorescent secondary antibody (AlexaFluor®488) for 50 min at 37°C. DAB was used to stain the nuclei. The slides were observed under a fluorescence microscope (Motic, Wetzlar, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to images the results at 200× magnification.

2.8 Statistical analysis

SPSS 22.0 was used for statistical analysis. The continuous data were expressed as mean ± standard deviation (SD) and were analyzed using Student’s t-test (two-group comparisons) or ANOVA with the LSD post hoc test (multiple group comparison). Categorical data were presented as n (%) and analyzed using the chi-square test. P-values <0.05 were considered statistically significant.

3 Results

3.1 Examination of the bone defects and the Masquelet technique model

X-ray at 4 weeks after surgery showed that the alignment of the osteotomy fracture in the observation group was good. The plates and screws were in place, and the stable 4-mm bone defect area was maintained (Fig 1A). For the PMMA implantation, the cement was in a normal position without loosening or overturning (Fig 1B).

Fig 1 — **Characterization of the bone defects** (A) X-ray of the typical models. X-ray showed good limb strength line, stable 4-mm bone defect, and no loosening of the screws and plates. (B) Hematoxylin & eosin staining comparing the membranes harvested from bone defects (control group) and beside the PMMA cement (model group).

The induced membranes showed many small and round cells near the bone cement. A rich vascular network was formed far from the bone cement and adjacent to the muscle, while fibrous tissue parallel to the PMMA bone cement block was formed in the middle area (Fig 1C).

3.2 RDTF improves ternary regulation related factors

Except for the EG group, the other groups had a more typical inductive membrane structure and more abundant vascular distribution (Fig 2A). To further explore the potential machanism of RDTF in Induced membrane formation, some related biomarkers were detected from protein and gene level respectively.

The immunohistochemistry results demonstrated that the expression of the CD31 protein in the EG group was weaker than that in the other groups (Fig 2B). The endomucin and PDGF-BB proteins were expressed in the induced membrane tissues of each group, but all of them were weakly positive. HMGB1 was expressed abundantly in the induced membrane tissue of each group.

PCR results showed that compared with the empty control group, the mRNA expression of CD31, VEGFA, HMGB1, and PDGF-BB in the IG, LG, MG, and HG were significantly increased. The expression of endomucin mRNA was not significantly increased, and the expression level was significantly increased only when with high-dose RDTF. After the use of RDTF, the ternary regulation-related factors VEGFA and HMGB1 tended to increase. After the use of RDTF, regardless of the dose, the expression of PDGF-BB mRNA was significantly increased compared with the IG group, while the expression of HMGB1 and endomucin significantly increased only when high doses of RDTF were used compared with those of IG. And VEGFA expression was significantly increased when the dose of RDTF reached the medium dose. The results of CD31 also showed a general trend of increased expression (Fig 3A). Similar results were observed at the protein level (Fig 3B).

Fig 3 — (A) mRNA expression (qRT-PCR) of HMGB1, endomucin, CD31, VEGFA, and PDGF-BB in the six groups (empty control group, EG; induced membrane group, IG; low dose RDTF group, LD; medium dose group, MG; high dose group, HG). (B) Protein expression of HMGB1, endomucin, CD31, VEGFA, and PDGF-BB in the six groups ^ΔP<0.05 according to the line. *P<0.05 vs. the EG group.

3.3 RDTF improves bone quality and blood vessels in the bone defect area

Six weeks after surgery, as presented in Fig 4, PMMA was radiopaque so that the bone substitute and the broken ends were visible. The defect in the IFG group showed continuous osteogenesis. Cortical bone was well reconstructed, and the marrow cavity had formed. The defect in the IMG group showed discontinuous osteogenesis. The bone marrow cavity was not fully formed, and the reconstructed bone cortex was weak, with discontinuous bone cortex still visible. Nothing grew in the critical-sized bone defects in the ECG group. The micro-CT cross-sections showed more obvious new bone formation in the IFG group than in the other two groups (Fig 5).

Fig 5 — Micro-CT of the typical models in the empty control (ECG), induced membrane (IMG), and flavonoid-treated (IFG) groups.

In the ECG group, there were fibers, muscles, and connective tissues, but only small amounts of bone tissue, while the amount of bone tissue was higher in the IMG and IFG (Fig 6A). IFG displayed the features of the osteoplastic stage, with homogeneous osteogenic distribution, while IMG displayed inflammatory cell infiltration, a small amount of proliferative connective tissue, and uneven osteogenesis.

Fig 6 — (A) Hematoxylin and eosin staining (top) and modified red O-solid green cartilage staining (bottom) of the bone defect tissue in the typical models in the empty control (ECG), induced membrane (IMG), and flavonoid-treated (IFG) groups. (B) Angiographic results of bone defect tissue in each group.

Angiography showed that the blood vessels in the ECG group were dissected, and no obvious blood vessels were in the defect area. A small number of new blood vessels were observed in the defect area of the IMG, with continuous but uneven distribution. The quality of the blood vessels formed in the IFG was better, and the local blood supply was more abundant (Fig 6B).

4 Discussion

Rhizoma drynariae (gusuibu) is a classic herb used to treat fractures in traditional Chinese medicine [19, 20], the main effective ingredient in Rhizoma drynariae is RDTF. CD31^hiEmcn^hi vessels induced by PDGF-BB secreted by osteoclast precursors, together with osteoblasts and osteoclasts, constitute the ternary regulatory mechanism of bone tissue reconstruction [16]. This study aimed to determine whether RDTF can promote bone remodeling and induce membrane growth in the rat Masquelet model and to explore its molecular mechanism based on the ternary regulation theory. The preliminary results showed that RDTF positively affected angiogenesis and bone reconstruction in the bone defect area and on factors involved in ternary regulation.

The formation of the induction membrane is a foreign body reaction of tissues after PMMA implantation, which is embodied by inflammation and edema reaction induced by multinucleated giant cells [27]. At 2 weeks after implantation of PMMA, the induction membrane was observed to have internal and external structures. The inner layer is a synovium-like epithelial cell layer, and the outer layer is mainly composed of fibroblasts, myofibroblasts, and collagen [15, 28]. Masquelet described the induced membrane as a 1 to 2mm thick and has a similar histologically characters to the synovial membrane, providing a microenvironment conducive to bone healing. It was also noted that the induction membrane could provide a very rich blood supply and prevent the soft tissues from growing into the bone defect area and the bone graft absorbed [29–31]. Induced membranes are formed in a complex biological environment in which multiple cytokine systems are involved [32–34]. As reported by Masquelet et al. [8] and Viateau et al. [35, 36], the induced membrane is like periosteum, which both contain and promote regenerative bone cells, growth factors, and neovascularization. Pelissier et al. [9] showed large numbers of capillaries in the outer layer of the induced membrane tissue at various stages, with an average diameter of about 1.2 mm. Tang et al. [37] demonstrated that Dll4/Notch1 signaling is negatively associated with the vessel density of the induced membrane.

In the process of bone repair, the induction membrane acts as a bed for the bone graft and the role of recruiting and chemotactic BMSCs. Wang et al. [38] revealed that the MDSCs of the induced membrane promoted the angiogenesis of endothelial cells through the expression of VEGFA, Ang2, and HIF-1α, which was upregulated by the activation of STAT3 signaling. Due to the affinity between microvessels and osteoprogenitor cells and osteoblasts, the invasion of osteoprogenitor cells into the lesion area in the process of bone repair is often accompanied by vascular growth. There is growing evidence that new blood vessels do more than just establishing local circulation to the new bone and supplying oxygen and nutrients, and they can also directly promote osteogenesis [4]. The expression of neovascularization-related genes in the induced membrane tissue after intervention with RDTF could be used as the basis for future research. This study will also help better understand the biological properties of the induced membrane, which will help create a better microenvironment for bone healing.

This study showed that RDTF had a certain effect on the angiogenesis of the bone defect area, and the number of new vessels in the IFG group was significantly higher than the other groups. X-ray and micro-CT also showed that the mineralization was better than in the other two groups. It could promote the formation of callus in the defect area. From a macroscopical perspective, RDTF in the Masquelet technique positively affects angiogenesis and osteogenesis of the bone defect area.

CD31 expression in endothelial cells is closely related to endothelial cell migration and neovascularization and is commonly used as a marker to detect neovascularization [15]. Gouron et al. [15] confirmed that the formation of new blood vessels was continuously maintained during the whole formation process of the inducing membrane. Cuthbert et al. [39] described that CD31 was expressed in the vascular lumen edge in the immunohistochemical results, confirming the existence of abundant blood vessels in the induced membrane tissue. The immunohistochemistry results in this study confirmed the results of Cuthbert et al. [39], and CD31 was shown in the vascular lumen edge of the induced membrane tissue, which was consistent with the results of western blotting and qRT-PCR.

Park-Windhol et al. [40] found that endomucin could regulate VEGFR2 activity and affect VEGF-induced endothelial cell migration, growth, and tube morphogenesis. This study suggests that endomucin is a potential regulator of angiogenesis and might provide new targeted therapies for vascular diseases. Different blood vessels could be identified by the immunohistochemistry of specific cells. Using this technique, Kusumbe et al. [41] found that endothelial cells in the endosteum were both strongly positive for CD31 and endomucin, while the venous sinus located in the diaphysis was only weakly positive for CD31 and weakly expressed endomucin. CD31^hiEmcn^hi endothelial cells and CD31^loEmcn^lo cells were significantly different in the suspension cells of long bone [41]. Therefore, the authors first proposed a new term for intracapsular microvessels: CD31^hiEmcn^hi vessels, also known as H-type vessels. Endomucin plays an important role in microangiogenesis, and if the immunohistochemistry results show strong CD31 and endomucin expression, the presence of H-type vessels may be suggested. The WB results in the present study confirmed the expression of PDGF-BB protein in the induced membrane tissue, so whether there were H-type blood vessels in the induced membrane tissue was explored. Still, the immunohistochemistry results showed that the expression of endomucin in the induced membrane was weak rather than strong positive, leading to the conclusion that there were no CD31^hiEmcn^hi vessels in the induced membrane.

This study has limitations. The promoting effects of RDTF on each growth factor were not completely consistent, which might be related to the small sample size of this study and induced membrane sampling operation. The results showed that the concentration of RDTF had a certain effect on the secretion of each growth factor. In this study we didn’t performed the experiment in vitro, in the further study we need to do it and to test more bio-markers, in this way we can have a better understanding the molecular mechanism of the ternary regulation theory.

In conclusion, the pathological, X-ray, western blot, and qRT-PCR results confirmed the secretion of VEGF, HMGB1, PDGF-BB, CD31, and endomucin in the induced membrane of the model group was significantly higher than that in the empty control group. Although the expected CD31^hiEmcn^hi vessels in the induction membrane were not observed, this study confirmed that RDTF could promote the secretion of angiogenic factors in the induced membrane. The specific mechanisms still need to be further studied.

Supporting information

S1 Fig. The timeline and design of the experimental and the operation procedure.

(JPG)

Click here for additional data file.^{(206.7KB, jpg)}

S2 Fig

(A) Self-made femoral plate and osteotomy guide plate for rats. (B) The 4-mm precision osteotomy was performed using the osteotomy guide plate to create a rat femoral defect model (panel A). The bone defect area was filled with PMMA cement (panel B). Four weeks after the primary operation, the induction of the bone membrane was observed (panel C). The induced membrane tissue was taken out (panel D).

(JPG)

Click here for additional data file.^{(615.1KB, jpg)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was funded by National Natural Science Foundation of China [No.81603640, No.81974575] and Natural Science Foundation of Guangdong Province [No. 20171206].The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Dumic-Cule I, Pecina M, Jelic M, Jankolija M, Popek I, Grgurevic L, et al. Biological aspects of segmental bone defects management. International Orthopaedics. 2015;39(5):1005–11. doi: 10.1007/s00264-015-2728-4 WOS:000353350700027. [DOI] [PubMed] [Google Scholar]
2.Kujala S, Raatikainen T, Ryhanen J, Kaarela O, Jalovaara P. Composite implant of native bovine bone morphogenetic protein (BMP), collagen carrier and biocoral in the treatment of resistant ulnar nonunions: report of five preliminary cases. Archives of Orthopaedic and Trauma Surgery. 2004;124(1):26–30. doi: 10.1007/s00402-003-0599-4 WOS:000188748900004. [DOI] [PubMed] [Google Scholar]
3.Koo KT, Polimeni G, Qahash M, Kim CK, Wikesjo UME. Periodontal repair in dogs: guided tissue regeneration enhances bone formation in sites implanted with a coral-derived calcium carbonate biomaterial. Journal of Clinical Periodontology. 2005;32(1):104–10. doi: 10.1111/j.1600-051X.2004.00632.x WOS:000226347200018. [DOI] [PubMed] [Google Scholar]
4.Ramasamy SK, Kusumbe AP, Adams RH. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Febs Journal. 2014;281:323–. WOS:000359666802199. doi: 10.1038/nature13146 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Aktuglu K, Gunay H, Alakbarov J. Monofocal bone transport technique for bone defects greater than 5 cm in tibia: our experience in a case series of 24 patients. Injury-International Journal of the Care of the Injured. 2016;47:S40–S6. doi: 10.1016/s0020-1383(16)30838-5 WOS:000395191600006. [DOI] [PubMed] [Google Scholar]
6.Jiang Y, Guo Y-F, Meng Y-K, Zhu L, Chen A-M. A report of a novel technique: The comprehensive fibular autograft with double metal locking plate fixation (cFALP) for refractory post-operative diaphyseal femur fracture non-union treatment. Injury-International Journal of the Care of the Injured. 2016;47(10):2307–11. doi: 10.1016/j.injury.2016.07.026 WOS:000388166500044. [DOI] [PubMed] [Google Scholar]
7.Zeng Z-K, Yuan L-M, Jiang P-P, Huang F. Treatment of 10 year humeral shaft nonunion with segment bony defect: a case report. International Journal of Clinical and Experimental Medicine. 2016;9(10):20242–6. WOS:000391260400179. [Google Scholar]
8.Masquelet AC, Fitoussi F, Begue T, Muller GP. [Reconstruction of the long bones by the induced membrane and spongy autograft]. Annales de chirurgie plastique et esthetique. 2000;45(3):346–53. Epub 2000/08/10. . [PubMed] [Google Scholar]
9.Pelissier P, Masquelet AC, Bareille R, Pelissier SM, Amedee J. Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration. Journal of orthopaedic research: official publication of the Orthopaedic Research Society. 2004;22(1):73–9. Epub 2003/12/06. doi: 10.1016/S0736-0266(03)00165-7 . [DOI] [PubMed] [Google Scholar]
10.Masquelet AC, Begue T. The concept of induced membrane for reconstruction of long bone defects. The Orthopedic clinics of North America. 2010;41(1):27–37; table of contents. Epub 2009/11/26. doi: 10.1016/j.ocl.2009.07.011 . [DOI] [PubMed] [Google Scholar]
11.Yin Q, Sun Z, Gu S. Progress of Masquelet technique to repair bone defect. Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery. 2013;27(10):1273–6. . [PubMed] [Google Scholar]
12.Morris R, Hossain M, Evans A, Pallister I. Induced membrane technique for treating tibial defects gives mixed results. The bone & joint journal. 2017;99-b(5):680–5. Epub 2017/04/30. doi: 10.1302/0301-620X.99B5.BJJ-2016-0694.R2 . [DOI] [PubMed] [Google Scholar]
13.Chadayammuri V, Hake M, Mauffrey C. Innovative strategies for the management of long bone infection: a review of the Masquelet technique. Patient safety in surgery. 2015;9:32–. doi: 10.1186/s13037-015-0079-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Gruber HE, Riley FE, Hoelscher GL, Bayoumi EM, Ingram JA, Ramp WK, et al. Osteogenic and chondrogenic potential of biomembrane cells from the PMMA-segmental defect rat model. Journal of Orthopaedic Research. 2012;30(8):1198–212. doi: 10.1002/jor.22047 WOS:000305515600003. [DOI] [PubMed] [Google Scholar]
15.Gouron R, Petit L, Boudot C, Six I, Brazier M, Kamel S, et al. Osteoclasts and their precursors are present in the induced-membrane during bone reconstruction using the Masquelet technique. Journal of Tissue Engineering and Regenerative Medicine. 2017;11(2):382–9. doi: 10.1002/term.1921 WOS:000396996200008. [DOI] [PubMed] [Google Scholar]
16.Xie H, Cui Z, Wang L, Xia Z, Hu Y, Xian L, et al. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis. Nature Medicine. 2014;20(11):1270–8. doi: 10.1038/nm.3668 WOS:000344724300016. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Wong RWK, Rabie ABM. Systemic effect of crude extract from rhizome of Drynaria fortunei on bone formation in mice. Phytotherapy Research. 2006;20(4):313–5. doi: 10.1002/ptr.1842 WOS:000236946700014. [DOI] [PubMed] [Google Scholar]
18.Lee Y-E, Liu H-C, Lin Y-L, Liu S-H, Yang R-S, Chen R-M. Drynaria fortunei J. Sm. improves the bone mass of ovariectomized rats through osteocalcin-involved endochondral ossification. Journal of Ethnopharmacology. 2014;158:94–101. doi: 10.1016/j.jep.2014.10.016 WOS:000348084600012. [DOI] [PubMed] [Google Scholar]
19.Yin F-m, Xiao L-b, Zhang Y. Research progress on Drynaria fortunei naringin on inflammation and bone activity. Zhongguo gu shang = China journal of orthopaedics and traumatology. 2015;28(2):182–6. . [PubMed] [Google Scholar]
20.Song S, Gao Z, Lei X, Niu Y, Zhang Y, Li C, et al. Total Flavonoids of Drynariae Rhizoma Prevent Bone Loss Induced by Hindlimb Unloading in Rats. Molecules. 2017;22(7). doi: 10.3390/molecules22071033 WOS:000406621300007. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Wang X-L, Wang N-L, Zhang Y, Gao H, Pang W-Y, Wong M-S, et al. Effects of eleven flavonoids from the osteoprotective fraction of Drynaria fortunei (KUNZE) J. SM. on osteoblastic proliferation using an osteoblast-like cell line. Chemical & Pharmaceutical Bulletin. 2008;56(1):46–51. doi: 10.1248/cpb.56.46 WOS:000252918900009. [DOI] [PubMed] [Google Scholar]
22.Li N, Whitaker C, Xu Z, Heggeness M, Yang S-Y. Therapeutic effects of naringin on degenerative human nucleus pulposus cells for discogenic low back pain. Spine Journal. 2016;16(10):1231–7. doi: 10.1016/j.spinee.2016.05.007 WOS:000386910400043. [DOI] [PubMed] [Google Scholar]
23.Jeong JC, Lee JW, Yoon CH, Lee YC, Chung KH, Kim MG, et al. Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells. Journal of Ethnopharmacology. 2005;96(3):489–95. doi: 10.1016/j.jep.2004.09.038 WOS:000226427600019. [DOI] [PubMed] [Google Scholar]
24.Hung T-Y, Chen T-L, Liao M-H, Ho W-P, Liu D-Z, Chuang W-C, et al. Drynaria fortunei J. Sm. promotes osteoblast maturation by inducing differentiation-related gene expression and protecting against oxidative stress-induced apoptotic insults. Journal of Ethnopharmacology. 2010;131(1):70–7. doi: 10.1016/j.jep.2010.05.063 WOS:000281325500009. [DOI] [PubMed] [Google Scholar]
25.Zhang X-T, Jiang H-J, Liang Z-R, He F-L, Liao X-Q, Ren Y-X, et al. Osteopractic total flavone promoting rat extra-articular tendon-bone healing through mTOR pathway. Zhongguo gu shang = China journal of orthopaedics and traumatology. 2018;31(3):248–53. doi: 10.3969/j.issn.1003-0034.2018.03.011 . [DOI] [PubMed] [Google Scholar]
26.Chen L-l, Lei L-h, Ding P-h, Tang Q, Wu Y-m. Osteogenic effect of Drynariae rhizoma extracts and Naringin on MC3T3-E1 cells and an induced rat alveolar bone resorption model. Archives of Oral Biology. 2011;56(12):1655–62. doi: 10.1016/j.archoralbio.2011.06.008 WOS:000298201900026. [DOI] [PubMed] [Google Scholar]
27.Wang X, Wei F, Luo F, Huang K, Xie Z. Induction of granulation tissue for the secretion of growth factors and the promotion of bone defect repair. Journal of Orthopaedic Surgery and Research. 2015;10. doi: 10.1186/s13018-015-0287-4 WOS:000361281800006. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Henrich D, Seebach C, Nau C, Basan S, Relja B, Wilhelm K, et al. Establishment and characterization of the Masquelet induced membrane technique in a rat femur critical-sized defect model. Journal of Tissue Engineering and Regenerative Medicine. 2016;10(10):E382–E96. doi: 10.1002/term.1826 WOS:000387786200016. [DOI] [PubMed] [Google Scholar]
29.Pelissier P, Martin D, Baudet J, Lepreux S, Masquelet AC. Behaviour of cancellous bone graft placed in induced membranes. British Journal of Plastic Surgery. 2002;55(7):596–8. doi: 10.1054/bjps.2002.3936 WOS:000178874500017. [DOI] [PubMed] [Google Scholar]
30.Powerski M, Maier B, Frank J, Marzi I. Treatment of severe osteitis after elastic intramedullary nailing of a radial bone shaft fracture by using cancellous bone graft in Masquelet technique in a 13-year-old adolescent girl. Journal of Pediatric Surgery. 2009;44(8):E17–E9. doi: 10.1016/j.jpedsurg.2009.04.039 WOS:000268780300045. [DOI] [PubMed] [Google Scholar]
31.Viateau V, Bensidhoum M, Guillemin G, Petite H, Hannouche D, Anagnostou F, et al. Use of the Induced Membrane Technique for Bone Tissue Engineering Purposes: Animal Studies. Orthopedic Clinics of North America. 2010;41(1):49–+. doi: 10.1016/j.ocl.2009.07.010 WOS:000278176400007. [DOI] [PubMed] [Google Scholar]
32.Hankenson KD, Dishowitz M, Gray C, Schenker M. Angiogenesis in bone regeneration. Injury-International Journal of the Care of the Injured. 2011;42(6):556–61. doi: 10.1016/j.injury.2011.03.035 WOS:000291245800003. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Jones E, Yang X. Mesenchymal stem cells and bone regeneration: Current status. Injury-International Journal of the Care of the Injured. 2011;42(6):562–8. doi: 10.1016/j.injury.2011.03.030 WOS:000291245800004. [DOI] [PubMed] [Google Scholar]
34.Marsell R, Einhorn TA. The biology of fracture healing. Injury-International Journal of the Care of the Injured. 2011;42(6):551–5. doi: 10.1016/j.injury.2011.03.031 WOS:000291245800002. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Viateau V, Guillemin G, Calando Y, Logeart D, Oudina K, Sedel L, et al. Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects: an ovine model. Veterinary surgery: VS. 2006;35(5):445–52. Epub 2006/07/18. doi: 10.1111/j.1532-950X.2006.00173.x . [DOI] [PubMed] [Google Scholar]
36.Viateau V, Guillemin G, Bousson V, Oudina K, Hannouche D, Sedel L, et al. Long-bone critical-size defects treated with tissue-engineered grafts: a study on sheep. Journal of orthopaedic research: official publication of the Orthopaedic Research Society. 2007;25(6):741–9. Epub 2007/02/24. doi: 10.1002/jor.20352 . [DOI] [PubMed] [Google Scholar]
37.Tang Q, Jin H, Tong M, Zheng G, Xie Z, Tang S, et al. Inhibition of Dll4/Notch1 pathway promotes angiogenesis of Masquelet’s induced membrane in rats. Experimental & molecular medicine. 2018;50(4):1–15. Epub 2018/04/21. doi: 10.1038/s12276-018-0062-9 ; PubMed Central PMCID: PMC5938037. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Wang W, Zuo R, Long H, Wang Y, Zhang Y, Sun C, et al. Advances in the Masquelet technique: Myeloid-derived suppressor cells promote angiogenesis in PMMA-induced membranes. Acta biomaterialia. 2020;108:223–36. Epub 2020/03/14. doi: 10.1016/j.actbio.2020.03.010 . [DOI] [PubMed] [Google Scholar]
39.Cuthbert RJ, Churchman SM, Tan HB, McGonagle D, Jones E, Giannoudis PV. Induced periosteum a complex cellular scaffold for the treatment of large bone defects. Bone. 2013;57(2):484–92. doi: 10.1016/j.bone.2013.08.009 WOS:000326848000022. [DOI] [PubMed] [Google Scholar]
40.Park-Windhol C, Ng YS, Yang J, Primo V, Saint-Geniez M, D’Amore PA. Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling. Scientific Reports. 2017;7. doi: 10.1038/s41598-017-16852-x WOS:000417353800007. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone (vol 507, pg 323, 2014). Nature. 2014;513(7519). doi: 10.1038/nature13720 WOS:000342075800048. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0278688.r001

Decision Letter 0

Selvaraj Vimalraj

12 Aug 2022

PONE-D-22-18003Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet techniquePLOS ONE

Dear Dr. Li,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 26 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Selvaraj Vimalraj

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

3. Thank you for stating the following in your Competing Interests section:

"The authors report no conflict of interest."

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state ""The authors have declared that no competing interests exist."", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

This information should be included in your cover letter; we will change the online submission form on your behalf.

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

Additional Editor Comments:

Carefully consider the reviewers' comments and suggested to submit a revised manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments

1. What is the rationale behind choosing oral administration of RDTF rather than other modes of drug administration or even any implantation modes?

2. Did the author carry out experiments on the study of in vitro cyto-compatibility of the drug in any means?

3. Did the author go through literatures having similar work performed in any other animal models?

4. Kindly check the spelling for Histological in page 16 (Line 188), Quantitative Page 17 (Line 196), Page 18 (Line 217), Page 18 (Line 236) and change it wherever applicable.

5. Justify the reason for selecting these particular concentrations of the drug for animal experiment? Why the author did not try with series of concentrations of the drug but ended up with one particular concentration for HG, MG, LG in animal study? Please justify.

6. The author has performed experiments on animal model to evaluate the levels of proteins and other biomarkers but failed to work on the extraction of individual bio-active flavonoid and its role in protein expression at in vitro cellular level. How would you validate that the present study with whole tuber flavonoid extract of drynaria gives enough stuff on ternary regulation of bone defect in animal model?

7. It is validated that Rhizoma drynaria contains HCN and did the author work on any procedures to remove it before extracting the total flavonoids.

8. Also, did the author check the anti-oxidant activity of the compound using in vitro biological assays specially to study the effect of oxidative stress markers?

9. In page 10, (line 64-66) the sentence “After 4 to 8 weeks a membrane surrounding the spacer was formation, then the second stage surgery was operated, including removal of the spacer and implant the bone grafts (citation).” can be changed to “After 4 to 8 weeks, a membrane surrounding the spacer was formed and then the second stage surgery was operated to remove the spacer for implantation of bone grafts (Include citation) “

10. The author has claimed that the autografts implanted are rat coccyx. Justify how did the author manages post-operative pain for the animal, risk of fracture at the donor site.

11. Is there any quantified data on mesenchymal stem cell (MSCs) production in induced membranes of IFGs/ECG/IMG with respect to normal bone marrow of their respective groups to validate the osteo-angiogenic potential

12. Did the author perform any experiments with induction membranes retrieved from reconstructed CSD femur model to quantify angiogenic and osteogenic bio-markers

13. Did the author validate any results on soft callus regeneration during bone remodeling with PMMA like how it is done after bone graft repair? Since this method includes long treatment period, did the author experimented on early bone healing phases during PMMA post-surgery?

14. The author has claimed that the induction membrane serves as periosteum like matrix to help in cell proliferation, growth factor production and signaling but the expected study did not prove the pro-angiogenic ability of IMGs individually. The gene and protein expression studies as mentioned in Fig 3A and 3B for VEGFA and CD31 showed minimal effect. Justify why PECAM and VEGFA expressions are hindered in IMGs than other groups.

Reviewer #2: The manuscript titled on " Ternary regulation mechanism of Rhizoma drynariae total flavonoids on inducedmembrane formation and bone remodeling in Masquelet technique" is an interesting work done by author. However, there are few questions need to be addressed in this study.

1) what is the rationale for choosing Rhizoma drynariaetotal flavonoids(RDTF) ? There are several flavonoids currently used for treating bone defect. How different from other study ?

2) Bone remodeling involves the secretion of bone markers such as ALP, OC etc. Do an author checked the expression of these other than VEGF and PDGF?

3) section 2.4-2.7 contains lot of spelling mistake in the title. For example, instead of histological author wrote ological analysys. It needs to be corrected prior to publication.

Fig.4 legends are not visible, poor resolution. Need a better resolution. In the RT-PCR, author didn't explain how did normalization done. And what method did you use to calculate mRNA expresssion ?

Do an author checked any of the protein expression such as VEGF and PDGF?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: DR. DHARUNYA GOVINDARAJAN

Reviewer #2: Yes: Arumugam Balasubramanian

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Dec 6;17(12):e0278688. doi: 10.1371/journal.pone.0278688.r002

Author response to Decision Letter 0

30 Oct 2022

Response to Reviewers 1

1.What is the rationale behind choosing oral administration of RDTF rather than other modes of drug administration or even any implantation modes?

RDTF is extracted from a traditional Chinese herb Rhizoma drynariae (Chinese name, Gusuibu), and it is dissolved well in water can be well absorbed by the gastrointestinal system, so we choose oral administration of RDTF rather than other modes of drug administration.

2. Did the author carry out experiments on the study of in vitro cyto-compatibility of the drug in any means?

Our groups have done some experiments on the study of in vitro cyto-compatibility of the drug

3. Did the author go through literatures having similar work performed in any other animal models?

Yes, some studies are focused on the osteoporosis models.

4. Kindly check the spelling for Histological in page 16 (Line 188), Quantitative Page 17 (Line 196), Page 18 (Line 217), Page 18 (Line 236) and change it wherever applicable.

Thanks for the remind of the details, we will revise them carefully.

The dose is designed according to the clinical effective dose, and the HG, MG, LG is according the human effective dosage, and conversion of human and animal administration.

In this study we just performed on the animal model, and do not perform in vitro. In the future study we will test in vitro cellular level.

7. It is validated that Rhizoma drynaria contains HCN and did the author work on any procedures to remove it before extracting the total flavonoids.

The total flavonoids are purchase from the company; we didn’t remove the HCN by ourselves.

8. Also, did the author check the anti-oxidant activity of the compound using in vitro biological assays specially to study the effect of oxidative stress markers?

This is a very good suggestion for the next step study. Anti-oxidant is also very important in the vessel formation, in this study we didn’t test the oxidative stress markers, but in the future study we can do it.

Thanks for the remind, will change it.

10. The author has claimed that the autografts implanted are rat coccyx. Justify how did the author manages post-operative pain for the animal, risk of fracture at the donor site.

We use the Meloxicam（0.2mg/kg）in the first 3 days after operative, carefully separate the tissand suture the donor site , no animals found fracture in this study.

In this study we didn’t test this, in future study we can test it.

12. Did the author perform any experiments with induction membranes retrieved from reconstructed CSD femur model to quantify angiogenic and osteogenic bio-markers

Yes. Our group have done some experiments to test the induction membranes angiogenic and osteogenic bio-markers.

This is a very important concern of the bone regeneration during the PMMA, in this study we didn’t do this, but it can be revolved in the further study.

The results showed in Fig 3A and 3B demonstrate that compare to empty control group (EG), the induced membrane group (IG) has higher level in CD31 and VEGFA, this suggest that the induced membrane may has a pro-angiogenic ability compare to control group. Because the RDTF has the effect on angiogenic, so in groups of the LG、MG、HG has a higher level of VEGFA and CD31.

Response to Reviewers 2

1) what is the rationale for choosing Rhizoma drynariaetotal flavonoids(RDTF) ? There are several flavonoids currently used for treating bone defect. How different from other study ?

2) Bone remodeling involves the secretion of bone markers such as ALP, OC etc. Do an author checked the expression of these other than VEGF and PDGF?

Yes, in this study we didn’t test the ALP, OC, in this study we focused on the biomarkers of the vessel’s and in future study we will test the relationship between the bone markers and the vessels biomarkers, this is a very important suggestion of the further study.

3) section 2.4-2.7 contains lot of spelling mistake in the title. For example, instead of histological author wrote ological analysys. It needs to be corrected prior to publication.

Fig.4 legends are not visible, poor resolution. Need a better resolution. In the RT-PCR, author didn't explain how did normalization done. And what method did you use to calculate mRNA expresssion ?Do an author checked any of the protein expression such as VEGF and PDGF?

Thanks for the remind, we will revise the details.

Thanks for the kindly remind of the details, we will revise it.

For the normalization and calculate has add in the methods, The expression of each gene was normalized to GAPDH expression by using the 2-ΔΔCt method.

In this study we just use the RT-PCR and Western-blot to test the biomarkers.

Attachment

Submitted filename: Response to Reviewers .docx

Click here for additional data file.^{(19.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0278688.r003

Decision Letter 1

Selvaraj Vimalraj

22 Nov 2022

Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique

PONE-D-22-18003R1

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Selvaraj Vimalraj

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Kindly check the spelling and grammatical error and make changes wherever applicable. The answers are satisfying and so the manuscript can be forwarded for publication.

All the best.

Reviewer #2: The author clearly addressed all the comments raised in this manuscript. Therefore the manuscript can be accepted for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: DR. DHARUNYA GOVINDARAJAN

Reviewer #2: No

**********

PLoS One. doi: 10.1371/journal.pone.0278688.r004

Acceptance letter

Selvaraj Vimalraj

24 Nov 2022

PONE-D-22-18003R1

Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique

Dear Dr. Li:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Selvaraj Vimalraj

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. The timeline and design of the experimental and the operation procedure.

(JPG)

Click here for additional data file.^{(206.7KB, jpg)}

S2 Fig

(JPG)

Click here for additional data file.^{(615.1KB, jpg)}

Attachment

Submitted filename: Response to Reviewers .docx

Click here for additional data file.^{(19.2KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0278688.ref001] 1.Dumic-Cule I, Pecina M, Jelic M, Jankolija M, Popek I, Grgurevic L, et al. Biological aspects of segmental bone defects management. International Orthopaedics. 2015;39(5):1005–11. doi: 10.1007/s00264-015-2728-4 WOS:000353350700027. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref002] 2.Kujala S, Raatikainen T, Ryhanen J, Kaarela O, Jalovaara P. Composite implant of native bovine bone morphogenetic protein (BMP), collagen carrier and biocoral in the treatment of resistant ulnar nonunions: report of five preliminary cases. Archives of Orthopaedic and Trauma Surgery. 2004;124(1):26–30. doi: 10.1007/s00402-003-0599-4 WOS:000188748900004. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref003] 3.Koo KT, Polimeni G, Qahash M, Kim CK, Wikesjo UME. Periodontal repair in dogs: guided tissue regeneration enhances bone formation in sites implanted with a coral-derived calcium carbonate biomaterial. Journal of Clinical Periodontology. 2005;32(1):104–10. doi: 10.1111/j.1600-051X.2004.00632.x WOS:000226347200018. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref004] 4.Ramasamy SK, Kusumbe AP, Adams RH. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Febs Journal. 2014;281:323–. WOS:000359666802199. doi: 10.1038/nature13146 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref005] 5.Aktuglu K, Gunay H, Alakbarov J. Monofocal bone transport technique for bone defects greater than 5 cm in tibia: our experience in a case series of 24 patients. Injury-International Journal of the Care of the Injured. 2016;47:S40–S6. doi: 10.1016/s0020-1383(16)30838-5 WOS:000395191600006. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref006] 6.Jiang Y, Guo Y-F, Meng Y-K, Zhu L, Chen A-M. A report of a novel technique: The comprehensive fibular autograft with double metal locking plate fixation (cFALP) for refractory post-operative diaphyseal femur fracture non-union treatment. Injury-International Journal of the Care of the Injured. 2016;47(10):2307–11. doi: 10.1016/j.injury.2016.07.026 WOS:000388166500044. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref007] 7.Zeng Z-K, Yuan L-M, Jiang P-P, Huang F. Treatment of 10 year humeral shaft nonunion with segment bony defect: a case report. International Journal of Clinical and Experimental Medicine. 2016;9(10):20242–6. WOS:000391260400179. [Google Scholar]

[pone.0278688.ref008] 8.Masquelet AC, Fitoussi F, Begue T, Muller GP. [Reconstruction of the long bones by the induced membrane and spongy autograft]. Annales de chirurgie plastique et esthetique. 2000;45(3):346–53. Epub 2000/08/10. . [PubMed] [Google Scholar]

[pone.0278688.ref009] 9.Pelissier P, Masquelet AC, Bareille R, Pelissier SM, Amedee J. Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration. Journal of orthopaedic research: official publication of the Orthopaedic Research Society. 2004;22(1):73–9. Epub 2003/12/06. doi: 10.1016/S0736-0266(03)00165-7 . [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref010] 10.Masquelet AC, Begue T. The concept of induced membrane for reconstruction of long bone defects. The Orthopedic clinics of North America. 2010;41(1):27–37; table of contents. Epub 2009/11/26. doi: 10.1016/j.ocl.2009.07.011 . [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref011] 11.Yin Q, Sun Z, Gu S. Progress of Masquelet technique to repair bone defect. Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery. 2013;27(10):1273–6. . [PubMed] [Google Scholar]

[pone.0278688.ref012] 12.Morris R, Hossain M, Evans A, Pallister I. Induced membrane technique for treating tibial defects gives mixed results. The bone & joint journal. 2017;99-b(5):680–5. Epub 2017/04/30. doi: 10.1302/0301-620X.99B5.BJJ-2016-0694.R2 . [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref013] 13.Chadayammuri V, Hake M, Mauffrey C. Innovative strategies for the management of long bone infection: a review of the Masquelet technique. Patient safety in surgery. 2015;9:32–. doi: 10.1186/s13037-015-0079-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref014] 14.Gruber HE, Riley FE, Hoelscher GL, Bayoumi EM, Ingram JA, Ramp WK, et al. Osteogenic and chondrogenic potential of biomembrane cells from the PMMA-segmental defect rat model. Journal of Orthopaedic Research. 2012;30(8):1198–212. doi: 10.1002/jor.22047 WOS:000305515600003. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref015] 15.Gouron R, Petit L, Boudot C, Six I, Brazier M, Kamel S, et al. Osteoclasts and their precursors are present in the induced-membrane during bone reconstruction using the Masquelet technique. Journal of Tissue Engineering and Regenerative Medicine. 2017;11(2):382–9. doi: 10.1002/term.1921 WOS:000396996200008. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref016] 16.Xie H, Cui Z, Wang L, Xia Z, Hu Y, Xian L, et al. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis. Nature Medicine. 2014;20(11):1270–8. doi: 10.1038/nm.3668 WOS:000344724300016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref017] 17.Wong RWK, Rabie ABM. Systemic effect of crude extract from rhizome of Drynaria fortunei on bone formation in mice. Phytotherapy Research. 2006;20(4):313–5. doi: 10.1002/ptr.1842 WOS:000236946700014. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref018] 18.Lee Y-E, Liu H-C, Lin Y-L, Liu S-H, Yang R-S, Chen R-M. Drynaria fortunei J. Sm. improves the bone mass of ovariectomized rats through osteocalcin-involved endochondral ossification. Journal of Ethnopharmacology. 2014;158:94–101. doi: 10.1016/j.jep.2014.10.016 WOS:000348084600012. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref019] 19.Yin F-m, Xiao L-b, Zhang Y. Research progress on Drynaria fortunei naringin on inflammation and bone activity. Zhongguo gu shang = China journal of orthopaedics and traumatology. 2015;28(2):182–6. . [PubMed] [Google Scholar]

[pone.0278688.ref020] 20.Song S, Gao Z, Lei X, Niu Y, Zhang Y, Li C, et al. Total Flavonoids of Drynariae Rhizoma Prevent Bone Loss Induced by Hindlimb Unloading in Rats. Molecules. 2017;22(7). doi: 10.3390/molecules22071033 WOS:000406621300007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref021] 21.Wang X-L, Wang N-L, Zhang Y, Gao H, Pang W-Y, Wong M-S, et al. Effects of eleven flavonoids from the osteoprotective fraction of Drynaria fortunei (KUNZE) J. SM. on osteoblastic proliferation using an osteoblast-like cell line. Chemical & Pharmaceutical Bulletin. 2008;56(1):46–51. doi: 10.1248/cpb.56.46 WOS:000252918900009. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref022] 22.Li N, Whitaker C, Xu Z, Heggeness M, Yang S-Y. Therapeutic effects of naringin on degenerative human nucleus pulposus cells for discogenic low back pain. Spine Journal. 2016;16(10):1231–7. doi: 10.1016/j.spinee.2016.05.007 WOS:000386910400043. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref023] 23.Jeong JC, Lee JW, Yoon CH, Lee YC, Chung KH, Kim MG, et al. Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells. Journal of Ethnopharmacology. 2005;96(3):489–95. doi: 10.1016/j.jep.2004.09.038 WOS:000226427600019. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref024] 24.Hung T-Y, Chen T-L, Liao M-H, Ho W-P, Liu D-Z, Chuang W-C, et al. Drynaria fortunei J. Sm. promotes osteoblast maturation by inducing differentiation-related gene expression and protecting against oxidative stress-induced apoptotic insults. Journal of Ethnopharmacology. 2010;131(1):70–7. doi: 10.1016/j.jep.2010.05.063 WOS:000281325500009. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref025] 25.Zhang X-T, Jiang H-J, Liang Z-R, He F-L, Liao X-Q, Ren Y-X, et al. Osteopractic total flavone promoting rat extra-articular tendon-bone healing through mTOR pathway. Zhongguo gu shang = China journal of orthopaedics and traumatology. 2018;31(3):248–53. doi: 10.3969/j.issn.1003-0034.2018.03.011 . [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref026] 26.Chen L-l, Lei L-h, Ding P-h, Tang Q, Wu Y-m. Osteogenic effect of Drynariae rhizoma extracts and Naringin on MC3T3-E1 cells and an induced rat alveolar bone resorption model. Archives of Oral Biology. 2011;56(12):1655–62. doi: 10.1016/j.archoralbio.2011.06.008 WOS:000298201900026. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref027] 27.Wang X, Wei F, Luo F, Huang K, Xie Z. Induction of granulation tissue for the secretion of growth factors and the promotion of bone defect repair. Journal of Orthopaedic Surgery and Research. 2015;10. doi: 10.1186/s13018-015-0287-4 WOS:000361281800006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref028] 28.Henrich D, Seebach C, Nau C, Basan S, Relja B, Wilhelm K, et al. Establishment and characterization of the Masquelet induced membrane technique in a rat femur critical-sized defect model. Journal of Tissue Engineering and Regenerative Medicine. 2016;10(10):E382–E96. doi: 10.1002/term.1826 WOS:000387786200016. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref029] 29.Pelissier P, Martin D, Baudet J, Lepreux S, Masquelet AC. Behaviour of cancellous bone graft placed in induced membranes. British Journal of Plastic Surgery. 2002;55(7):596–8. doi: 10.1054/bjps.2002.3936 WOS:000178874500017. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref030] 30.Powerski M, Maier B, Frank J, Marzi I. Treatment of severe osteitis after elastic intramedullary nailing of a radial bone shaft fracture by using cancellous bone graft in Masquelet technique in a 13-year-old adolescent girl. Journal of Pediatric Surgery. 2009;44(8):E17–E9. doi: 10.1016/j.jpedsurg.2009.04.039 WOS:000268780300045. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref031] 31.Viateau V, Bensidhoum M, Guillemin G, Petite H, Hannouche D, Anagnostou F, et al. Use of the Induced Membrane Technique for Bone Tissue Engineering Purposes: Animal Studies. Orthopedic Clinics of North America. 2010;41(1):49–+. doi: 10.1016/j.ocl.2009.07.010 WOS:000278176400007. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref032] 32.Hankenson KD, Dishowitz M, Gray C, Schenker M. Angiogenesis in bone regeneration. Injury-International Journal of the Care of the Injured. 2011;42(6):556–61. doi: 10.1016/j.injury.2011.03.035 WOS:000291245800003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref033] 33.Jones E, Yang X. Mesenchymal stem cells and bone regeneration: Current status. Injury-International Journal of the Care of the Injured. 2011;42(6):562–8. doi: 10.1016/j.injury.2011.03.030 WOS:000291245800004. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref034] 34.Marsell R, Einhorn TA. The biology of fracture healing. Injury-International Journal of the Care of the Injured. 2011;42(6):551–5. doi: 10.1016/j.injury.2011.03.031 WOS:000291245800002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref035] 35.Viateau V, Guillemin G, Calando Y, Logeart D, Oudina K, Sedel L, et al. Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects: an ovine model. Veterinary surgery: VS. 2006;35(5):445–52. Epub 2006/07/18. doi: 10.1111/j.1532-950X.2006.00173.x . [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref036] 36.Viateau V, Guillemin G, Bousson V, Oudina K, Hannouche D, Sedel L, et al. Long-bone critical-size defects treated with tissue-engineered grafts: a study on sheep. Journal of orthopaedic research: official publication of the Orthopaedic Research Society. 2007;25(6):741–9. Epub 2007/02/24. doi: 10.1002/jor.20352 . [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref037] 37.Tang Q, Jin H, Tong M, Zheng G, Xie Z, Tang S, et al. Inhibition of Dll4/Notch1 pathway promotes angiogenesis of Masquelet’s induced membrane in rats. Experimental & molecular medicine. 2018;50(4):1–15. Epub 2018/04/21. doi: 10.1038/s12276-018-0062-9 ; PubMed Central PMCID: PMC5938037. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref038] 38.Wang W, Zuo R, Long H, Wang Y, Zhang Y, Sun C, et al. Advances in the Masquelet technique: Myeloid-derived suppressor cells promote angiogenesis in PMMA-induced membranes. Acta biomaterialia. 2020;108:223–36. Epub 2020/03/14. doi: 10.1016/j.actbio.2020.03.010 . [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref039] 39.Cuthbert RJ, Churchman SM, Tan HB, McGonagle D, Jones E, Giannoudis PV. Induced periosteum a complex cellular scaffold for the treatment of large bone defects. Bone. 2013;57(2):484–92. doi: 10.1016/j.bone.2013.08.009 WOS:000326848000022. [DOI] [PubMed] [Google Scholar]

[pone.0278688.ref040] 40.Park-Windhol C, Ng YS, Yang J, Primo V, Saint-Geniez M, D’Amore PA. Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling. Scientific Reports. 2017;7. doi: 10.1038/s41598-017-16852-x WOS:000417353800007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278688.ref041] 41.Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone (vol 507, pg 323, 2014). Nature. 2014;513(7519). doi: 10.1038/nature13720 WOS:000342075800048. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique

Ding Li

Dun Zhao

Zhikui Zeng

Feng Huang

Ziwei Jiang

Hao Xiong

Tianan Guan

Bin Fang

Yue Li

Roles

Abstract

Context

Objective

Methods

Results

Discussion and conclusion

1 Introduction

2 Material and methods

2.1 Animals and Masquelet’s technique model establishment

2.2 Treatment

2.3 Micro CT and angiography analyses

2.4 Histoological analysis

2.5 Quantitative real-time polymerase chain reaction (qRT-PCR)

2.6 Western-blot

2.7 Immunohistochemistry

2.8 Statistical analysis

3 Results

3.1 Examination of the bone defects and the Masquelet technique model

Fig 1.

3.2 RDTF improves ternary regulation related factors

Fig 2. Histological examination and immunohistochemistry in the five groups (empty control group, EG; induced membrane group, IG; low dose RDTF group, LD; medium dose group, MG; high dose group, HG).

Fig 3. Expression of genes involved in the ternary regulation of bone defect repair.

3.3 RDTF improves bone quality and blood vessels in the bone defect area

Fig 4. X-Ray in the bone defect area.

Fig 5. Micro-computed tomography in the bone defect area.

Fig 6. Effects of RDTF on bone histological examination in the bone defect area.

4 Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Selvaraj Vimalraj

Roles

Author response to Decision Letter 0

Decision Letter 1

Selvaraj Vimalraj

Roles

Acceptance letter

Selvaraj Vimalraj

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases