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Cottrill E, Rajkovic CJ, Lin J, Shafi M, Tracz JA, Hernandez V, Parhami F, Witham TF. Synthesis and Preclinical Evaluation of a Novel Oxy133-Infused Biomimetic Bone Graft Using a Rat Model of Posterolateral Spinal Fusion. World Neurosurg 2025; 194:123551. [PMID: 39653079 DOI: 10.1016/j.wneu.2024.12.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2024] [Accepted: 12/03/2024] [Indexed: 01/07/2025]
Abstract
OBJECTIVE To 1) create a novel tissue-engineered bone graft comprising the osteoinductive oxysterol Oxy133 and 2) compare the osteogenic capability of this novel bone graft with bone graft substitutes previously examined. METHODS Oxy133 was homogeneously incorporated into a biomimetic (BioMim) bone graft substitute comprising extracellular matrix and calcium phosphates. Two iterations of the graft were created: one corresponding to an implant-dose of 2.0 mg Oxy133 (BioMim-Oxy133-Lo) and the other corresponding to an implant-dose of 20 mg Oxy133 (BioMim-Oxy133-Hi). Thirty-two male Sprague-Dawley rats were allocated randomly to 4 equally sized groups: 1) BioMim-Oxy133-Lo, 2) BioMim-Oxy133-Hi, 3) absorbable collagen sponge (ACS) with topically applied Oxy133 dissolved in dimethyl sulfoxide (ACS-Oxy133; 20 mg Oxy133/graft), and 4) ACS with topically applied recombinant human bone morphogenetic protein-2 (rhBMP-2) dissolved in water (ACS-rhBMP-2; 5.0 μg rhBMP-2/graft). All animals underwent L4-L5 posterolateral spinal fusion. Spines were harvested 8 weeks postoperatively and analyzed using micro-computed tomography imaging. RESULTS Successful fusion was achieved in all animals. Grafts containing Oxy133 had significantly greater bone volume, percent of bone volume per tissue volume (%BV), bone surface density (BSD), and trabecular number (TbN) compared to ACS-rhBMP-2 (P < 0.01 for each). Animals treated with BioMim-Oxy133-Lo had the greatest %BV, BSD, and TbN (P < 0.001 for each), whereas animals treated with ACS-rhBMP-2 had the lowest %BV, BSD, TbN, and trabecular thickness (P < 0.001 for each). CONCLUSIONS BioMim-Oxy133 is a novel bone graft that led to superior bone volume and quality compared to ACS-rhBMP-2 in a clinically translatable rat model of spinal fusion. Future work is needed to further evaluate this material as a safe and efficacious bone graft substitute.
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Affiliation(s)
- Ethan Cottrill
- The Spinal Fusion Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Orthopedic Surgery, Duke University School of Medicine, Durham, North Carolina, USA.
| | - Christian J Rajkovic
- The Spinal Fusion Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Jessica Lin
- The Spinal Fusion Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Mahnoor Shafi
- The Spinal Fusion Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Jovanna A Tracz
- The Spinal Fusion Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Vaughn Hernandez
- The Spinal Fusion Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | | | - Timothy F Witham
- The Spinal Fusion Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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Lin CL, Chen YW, Kuo CH, Tu TY, Wu HL, Tsai JC, Shyong YJ. Calcium phosphate complex of recombinant human thrombomodulin promote bone formation in interbody fusion. Biofabrication 2024; 17:015010. [PMID: 39326445 DOI: 10.1088/1758-5090/ad8035] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2024] [Accepted: 09/26/2024] [Indexed: 09/28/2024]
Abstract
Interbody fusion is an orthopedic surgical procedure to connect two adjacent vertebrae in patients suffering from spinal disc disease. The combination of synthetic bone grafts with protein-based drugs is an intriguing approach to stimulate interbody bone growth, specifically in patients exhibiting restricted bone progression. Recombinant human thrombomodulin (rhTM), a novel protein drug characterized by its superior stability and potency, shows promise in enhancing bone formation. A composite bone graft, termed CaP-rhTM, has been synthesized, combining calcium phosphate (CaP) microparticles as a delivery vehicle for rhTM to facilitate interbody fusion.In vitrostudies have demonstrated that rhTM significantly promotes the proliferation and maturation of preosteoblasts at nanogram dosage, while exerting minimal impact on osteosarcoma cell growth. The expression levels of mature osteoblast markers, including osteocalcin, osteopontin, alkaline phosphatase, and calcium deposition were also enhanced by rhTM. In rat caudal disc model of interbody fusion, CaP-rhTM with 800 ng of drug dosage was implanted along with a polylactic acid cage, to ensure structural stability within the intervertebral space. Microcomputed tomography analyses revealed that from 8 to 24 weeks, CaP-rhTM substantially improves both bone volume and trabecular architecture, in addition to the textural integrity of bony endplate surfaces. Histological examination confirmed the formation of a continuous bone bridge connecting adjacent vertebrae. Furthermore, biomechanical assessment via three-point bending tests indicated an improved bone quality of the fused disc. This study has demostrated that rhTM exhibits considerable potential in promoting osteogenesis. The use of CaP-rhTM has also shown significant improvements in promoting interbody fusion.
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Affiliation(s)
- Cheng-Li Lin
- Department of Orthopedic Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, No.138, Sheng Li Road, Tainan 704, Taiwan (R.O.C)
| | - Yu-Wei Chen
- School of Pharmacy, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan (R.O.C)
| | - Cheng-Hsiang Kuo
- International Center for Wound Repair and Regeneration, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan (R.O.C)
| | - Ting-Yuan Tu
- Department of Biomedical Engineering, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan (R.O.C)
| | - Hua-Lin Wu
- Department of Biochemistry and Molecular Biology, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan (R.O.C)
| | - Jui-Chen Tsai
- School of Pharmacy, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan (R.O.C)
| | - Yan-Jye Shyong
- School of Pharmacy, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan (R.O.C)
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Broussolle T, Roux JP, Chapurlat R, Barrey C. Murine models of posterolateral spinal fusion: A systematic review. Neurochirurgie 2023; 69:101428. [PMID: 36871885 DOI: 10.1016/j.neuchi.2023.101428] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 02/05/2023] [Accepted: 02/07/2023] [Indexed: 03/07/2023]
Abstract
BACKGROUND Rodent models are commonly used experimentally to assess treatment effectiveness in spinal fusion. Certain factors are associated with better fusion rates. The objectives of the present study were to report the protocols most frequently used, to evaluate factors known to positively influence fusion rate, and to identify new factors. METHOD A systematic literature search of PubMed and Web of Science found 139 experimental studies of posterolateral lumbar spinal fusion in rodent models. Data for level and location of fusion, animal strain, sex, weight and age, graft, decortication, fusion assessment and fusion and mortality rates were collected and analyzed. RESULTS The standard murine model for spinal fusion was male Sprague Dawley rats of 295g weight and 13 weeks' age, using decortication, with L4-L5 as fusion level. The last two criteria were associated with significantly better fusion rates. On manual palpation, the overall mean fusion rate in rats was 58% and the autograft mean fusion rate was 61%. Most studies evaluated fusion as a binary on manual palpation, and only a few used CT and histology. Average mortality was 3.03% in rats and 1.56% in mice. CONCLUSIONS These results suggest using a rat model, younger than 10 weeks and weighing more than 300 grams on the day of surgery, to optimize fusion rates, with decortication before grafting and fusing the L4-L5 level.
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Affiliation(s)
- T Broussolle
- Department of Spine Surgery, P. Wertheimer University Hospital, GHE, hospices civils de Lyon, université Claude-Bernard Lyon 1, Lyon, France; Inserm UMR 1033, université Claude-Bernard Lyon 1, Lyon, France.
| | - Jean-Paul Roux
- Inserm UMR 1033, université Claude-Bernard Lyon 1, Lyon, France
| | - R Chapurlat
- Inserm UMR 1033, université Claude-Bernard Lyon 1, Lyon, France
| | - C Barrey
- Department of Spine Surgery, P. Wertheimer University Hospital, GHE, hospices civils de Lyon, université Claude-Bernard Lyon 1, Lyon, France; Arts et métiers ParisTech, ENSAM, 151, boulevard de l'Hôpital, 75013 Paris, France
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Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro. Int J Mol Sci 2021; 22:ijms22063163. [PMID: 33808940 PMCID: PMC8003796 DOI: 10.3390/ijms22063163] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Revised: 03/14/2021] [Accepted: 03/15/2021] [Indexed: 12/11/2022] Open
Abstract
The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.
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Cottrill E, Lazzari J, Pennington Z, Ehresman J, Schilling A, Dirckx N, Theodore N, Sciubba D, Witham T. Oxysterols as promising small molecules for bone tissue engineering: Systematic review. World J Orthop 2020; 11:328-344. [PMID: 32908817 PMCID: PMC7453739 DOI: 10.5312/wjo.v11.i7.328] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Revised: 05/08/2020] [Accepted: 07/01/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Bone tissue engineering is an area of continued interest within orthopaedic surgery, as it promises to create implantable bone substitute materials that obviate the need for autologous bone graft. Recently, oxysterols – oxygenated derivatives of cholesterol – have been proposed as a novel class of osteoinductive small molecules for bone tissue engineering. Here, we present the first systematic review of the in vivo evidence describing the potential therapeutic utility of oxysterols for bone tissue engineering.
AIM To systematically review the available literature examining the effect of oxysterols on in vivo bone formation.
METHODS We conducted a systematic review of the literature following PRISMA guidelines. Using the PubMed/MEDLINE, Embase, and Web of Science databases, we queried all publications in the English-language literature investigating the effect of oxysterols on in vivo bone formation. Articles were screened for eligibility using PICOS criteria and assessed for potential bias using an expanded version of the SYRCLE Risk of Bias assessment tool. All full-text articles examining the effect of oxysterols on in vivo bone formation were included. Extracted data included: Animal species, surgical/defect model, description of therapeutic and control treatments, and method for assessing bone growth. Primary outcome was fusion rate for spinal fusion models and percent bone regeneration for critical-sized defect models. Data were tabulated and described by both surgical/defect model and oxysterol employed. Additionally, data from all included studies were aggregated to posit the mechanism by which oxysterols may mediate in vivo bone formation.
RESULTS Our search identified 267 unique articles, of which 27 underwent full-text review. Thirteen studies (all preclinical) met our inclusion/exclusion criteria. Of the 13 included studies, 5 employed spinal fusion models, 2 employed critical-sized alveolar defect models, and 6 employed critical-sized calvarial defect models. Based upon SYRCLE criteria, the included studies were found to possess an overall “unclear risk of bias”; 54% of studies reported treatment randomization and 38% reported blinding at any level. Overall, seven unique oxysterols were evaluated: 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, Oxy4/Oxy34, Oxy18, Oxy21/Oxy133, and Oxy49. All had statistically significant in vivo osteoinductive properties, with Oxy4/Oxy34, Oxy21/Oxy133, and Oxy49 showing a dose-dependent effect in some cases. In the eight studies that directly compared oxysterols to rhBMP-2-treated animals, similar rates of bone growth occurred in the two groups. Biochemical investigation of these effects suggests that they may be primarily mediated by direct activation of Smoothened in the Hedgehog signaling pathway.
CONCLUSION Present preclinical evidence suggests oxysterols significantly augment in vivo bone formation. However, clinical trials are necessary to determine which have the greatest therapeutic potential for orthopaedic surgery patients.
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Affiliation(s)
- Ethan Cottrill
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Julianna Lazzari
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Zach Pennington
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Jeff Ehresman
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Andrew Schilling
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Naomi Dirckx
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Nicholas Theodore
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Daniel Sciubba
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
| | - Timothy Witham
- Department of Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States
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Çetin E, Daldal İ, Eren A, Akarca Dizakar SÖ, Ömeroğlu S, Uzuner B, Çelik HH, Saygılı HH, Koçkar B, Şenköylü A. Epidermal growth factor enhances spinal fusion: Posterolateral lumbar fusion model on rats. ACTA ORTHOPAEDICA ET TRAUMATOLOGICA TURCICA 2019; 53:134-139. [PMID: 30738625 PMCID: PMC6506815 DOI: 10.1016/j.aott.2019.01.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Revised: 11/30/2018] [Accepted: 01/17/2019] [Indexed: 12/31/2022]
Abstract
Objective The aim of this study was to investigate the effects of human recombinant epidermal growth factor (EGF) on posterolateral lumbar fusion in a rat model. Methods 36 male Sprague Dawley rats underwent posterolateral fusion at L4-5 level. They were randomly assigned to 3 groups: 1- Sham control group where no local augmentation was made, 2- Local Hydoxyapatite β-tricalcium phosphate (HA/β-TCP) augmentation group and 3- Local HA/β-TCP + EGF augmentation group. Rats were euthanized at 8 weeks post-surgery. 6 rats from each group were selected for manual palpation examination, micro-computed tomography analysis and histologic analysis; and the rest was used for biomechanical analysis. Results Based on manual palpation, there was no fusion in the sham control group. Fusion rate was 33.3% in the HA/β-TCP group and 66.7% in the HA/β-TCP + EGF group (p = 0.085). Micro-CT results revealed that new bone formation was higher in the HA/β-TCP + EGF group (BV/TV: 40% vs. 65%) (p = 0.004). Histologically newly formed bone tissue was more pronounced in the EGF group and compacted and bridging bone spicules were observed. The median maximum bending moment values were 0.51 Nmm (0.42–0.59), 0.73 Nmm (0.49–0.88) and 0.91 Nmm (0.66–1.03) in the sham control, HA/β-TCP and HA/β-TCP + EGF groups, respectively (p = 0.013). The median stiffness values were 1.69 N/mm (1.12–2.18), 1.68 N/mm (1.13–2.74) and 3.10 N/mm (1.66–4.40) as in the previous order (p = 0.087). Conclusion This study demonstrates that EGF enhances posterolateral lumbar fusion in the rat model. EGF in combination with ceramic grafts increased the fusion rates. Our findings may provide insights to further studies, investigating EGF's clinical usage as an alternative fusion enhancer.
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Affiliation(s)
- Engin Çetin
- Department of Orthopaedics and Traumatology, Gaziosmanpaşa Taksim Training and Research Hospital, Istanbul, Turkey.
| | - İsmail Daldal
- Department of Orthopaedics and Traumatology, Gazi University Faculty of Medicine, Ankara, Turkey.
| | - Ali Eren
- Department of Orthopaedics and Traumatology, Gazi University Faculty of Medicine, Ankara, Turkey.
| | | | - Suna Ömeroğlu
- Department of Histology and Embryology, Gazi University Faculty of Medicine, Ankara, Turkey.
| | - Bora Uzuner
- Department of Anatomy, Hacettepe University Faculty of Medicine, Ankara, Turkey.
| | - Hakan Hamdi Çelik
- Department of Anatomy, Hacettepe University Faculty of Medicine, Ankara, Turkey.
| | | | - Benat Koçkar
- Department of Mechanical Engineering, Hacettepe University, Ankara, Turkey.
| | - Alpaslan Şenköylü
- Department of Orthopaedics and Traumatology, Gazi University Faculty of Medicine, Ankara, Turkey.
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Lee JS, Kim E, Han S, Kang KL, Heo JS. Evaluating the oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol in periodontal regeneration using periodontal ligament stem cells and alveolar bone healing models. Stem Cell Res Ther 2017; 8:276. [PMID: 29208033 PMCID: PMC5717822 DOI: 10.1186/s13287-017-0725-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2017] [Revised: 11/03/2017] [Accepted: 11/13/2017] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Oxysterols, oxygenated by-products of cholesterol biosynthesis, play roles in various physiological and pathological systems. However, the effects of oxysterols on periodontal regeneration are unknown. This study investigated the effects of the specific oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on the regeneration of periodontal tissues using in-vitro periodontal ligament stem cells (PDLSCs) and in-vivo models of alveolar bone defect. METHODS To evaluate the effects of the combined oxysterols on PDLSC biology, we studied the SS-induced osteogenic differentiation of PDLSCs by assessing alkaline phosphatase activity, intracellular calcium levels [Ca2+]i, matrix mineralization, and osteogenic marker mRNA expression and protein levels. To verify the effect of oxysterols on alveolar bone regeneration, we employed tooth extraction bone defect models. RESULTS Oxysterols increased the osteogenic activity of PDLSCs compared with the control group. The expression of liver X receptor (LXR) α and β, the nuclear receptors for oxysterols, and their target gene, ATP-binding cassette transporter A1 (ABCA1), increased significantly during osteogenesis. Oxysterols also increased protein levels of the hedgehog (Hh) receptor Smo and the transcription factor Gli1. We further confirmed the reciprocal reaction between the LXRs and Hh signaling. Transfection of both LXRα and LXRβ siRNAs decreased Smo and Gli1 protein levels. In contrast, the inhibition of Hh signaling attenuated the LXRα and LXRβ protein levels. Subsequently, SS-induced osteogenic activity of PDLSCs was suppressed by the inhibition of LXRs or Hh signaling. The application of SS also enhanced bone formation in the defect sites of in-vivo models, showing equivalent efficacy to recombinant human bone morphogenetic protein-2. CONCLUSIONS These findings suggest that a specific combination of oxysterols promoted periodontal regeneration by regulating PDLSC activity and alveolar bone regeneration.
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Affiliation(s)
- Jin-Sun Lee
- Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul, 02447, South Korea
| | - EunJi Kim
- Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul, 02447, South Korea
| | - Seonggu Han
- Department of Periodontology, School of Dentistry, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul, 02447, South Korea
| | - Kyung Lhi Kang
- Department of Periodontology, School of Dentistry, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul, 02447, South Korea.
| | - Jung Sun Heo
- Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul, 02447, South Korea.
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