• Title/Summary/Keyword: immunohistochemical methods

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THE EXPERIMENTAL STUDY OF THE BONE REGENERATION ON RABBIT MAXILLARY SINUS GRAFTING WITH ${\beta}$-TCP (가토 상악동에 이식된 ${\beta}$-TCP의 골치유에 관한 실험적 연구)

  • Park, Jung-Ha;Hwang, Kyung-Gyun;Park, Chang-Joo;Choi, Yong-Soo;Ma, Pyung-Soo;Paik, Seung-Sam;Shim, Kwang-Sup
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.32 no.2
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    • pp.107-116
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    • 2006
  • Purpose:Maxillay sinus grafting is an effective treatment procedure to improve bone height in the posterior maxillar area for implant installation. Beta-tricalciumphosphate(${\beta}$-TCP) was introduced to be grafting substitute material, providing a reasonable bio-degradation time, no need for harvesting procedure. The purpose of this study is to evaluate bone healing & regeneration phase using histomorphometric and immunohistochemical analysis. Material & Methods:Sixteen rabbits were divided into 4 groups. Bi-lateral maxillary sinus membranes were elevated at each rabbits, ${\beta}$-TCP was augmented in left sinus, autogenous bone was augmented in right sinus. The rabbits were sacrificed at 2, 4, 8 and 12 weeks. We investigated the bone regeneration & growth factor expression. Results: 1. The mean new bone volume formation was 28.99${\pm}$6.55%, 49.54${\pm}$5.47%, 69.09${\pm}$8.90% in autogenous grafted area, and 22.86${\pm}$5.56%, 24.00${\pm}$4.09%, 34.11${\pm}$3.37% in ${\beta}$-TCP area at 4, 8, 12 weeks. Therefore, new bone formation in autogenous bone was significantly higher than ${\beta}$-TCP (p<0.05). 2. The BMP 2/4 expression in autogenous bone grafted area was higher at 4, 8 weeks. 3. There was no difference in expression pattern of BMP-7/PDGF/VEGF during grafted bone regeneration. Conclusion:The authors we conclude that the autogenous bone graft was faster than ${\beta}$-TCP in bone regeneration, and the BMP 2/4 were more important in graft bone regeneration.

Immunohistochemical Study on Hair Growth Promoting Effect of Yonnyuniksoogobon-dan (연년익수고본단(延年益壽固本丹)의 육모효능(育毛效能)에 대한 면역조직화학적 연구)

  • Jeong, Chun-Geun;Shim, Eun-Sheb;Lee, Chang-Yeol;Kim, Bum-Hoi;Kim, Seong-Joon;Kang, Hee;Sohn, Nak-Won
    • The Journal of Korean Medicine
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    • v.31 no.2
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    • pp.48-63
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    • 2010
  • Objectives: Yonnyuniksoogobon-dan (Yan Nian Yi Shou Gu Ben Dan 延年益壽固本丹) is composed of 11 herbs (Polygoni Mutiflori Radix, Lycii Radicis Cortex, Polia, Rehmanniae Radix, Rehmanniae Radix Preparat, Asparagi Radix, Liriopis Tuber, Lycii Fructus, Acori Graminei Rhizoma, Angelicae Acutiloba Radix, and Pini Folium) based on Yonryunggobon-dan (Yan Ling Gu Ben Dan 延齡固本丹) and Yonnyuniksoobulrho-dan (Yan Nian Yi Shou Bu Lao Dan 延年益壽不老丹). This study evaluated hair growth promoting effect of Yonnyuniksoogobon-dan on the shaved C57BL/6 mice. Methods: Yonnyuniksoogobon-dan was treated by oral administration (Sample I) and oral administration plus skin application (Sample II) once a day for 12 days. Hair regrowth was photographically and histologically determined during the experimental period. Hair growth cycle related factors (EGF, TGF-${\beta}1$) and vascular factors (VEGF, iNOS) were also determined with immunohistochemistry. Results: 1. On gross observation of hair regrowth, Sample I and Sample II groups demonstrated acceleration of hair regrowth. 2. The hair regrowth index of the Sample I group increased significantly from 7 days (P<0.05) to 12 days (P<0.01) after the shave while that of the Sample II group significantly increased at 12 days (P<0.05). 3. On histological observation, both Sample I and Sample II groups demonstrated histological improvement and increases of number and diameter of the hair follicles. 4. EGF expressions on the root sheath of hair follicles were up-regulated in both Sample I and Sample II groups. 5. TGF-${\beta}1$ expressions on the root sheath of hair follicles were not regulated in Sample I or Sample II groups. 6. VEGF expressions in the surrounding tissues of hair follicles were up-regulated in both Sample I and Sample II groups. 7. iNOS expressions in the surrounding tissues of hair follicles were down-regulated in both Sample I and Sample II groups. Conclusions: These results suggest that Yonnyuniksoogobon-dan has hair growth-promoting activity and these effects relate to up-regulations of EGF and VEFG expressions and down-regulations of TGF-${\beta}1$ and iNOS expressions on hair roots.

Immunohistochemical Study on the Hair Growth Promoting Effect of Yonnyuniksoogobon-dan

  • Jeong, Chun-Geun;Park, Min-Hee;Seong, Ju-Won;Lee, Hyun-Sam;Park, Seong-Kyu;Kim, Sun-Yeou;Kim, Yoon-Bum;Jung, Hyuk-Sang;Sohn, Nak-Won;Sohn, Young-Joo
    • The Journal of Korean Medicine
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    • v.29 no.5
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    • pp.77-89
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    • 2008
  • Objectives : This study evaluates the hair growth promoting effect of Yonnyuniksoogobon-dan on shaved C57BL6 mice. Methods : Yonnyuniksoogobon-dan was administered orally (Group I) and both orally and by skin application (Group II) once a day for 12 days. The experimental groups were compared to Control, which was orally administered physiological saline solution. Hair regrowth was photographically and histologically determined during the experimental period. The levels of hair growth cycle related factors (EGF, TGF-${\beta}$1) and vascular factors (VEGF, iNOS) were also determined by immunohistochemistry. On gross observation of hair growth, both Group I and Group II shaved C57BL6 mice showed accelerated hair regrowth. Results : The hair regrowth index of Group I increased significantly from 7 days (P<0.05) to 12 days (P<0.01) after shaving and that of Group II was significantly higher at 12 days (P<0.05). On histological observation, both Group I and Group II demonstrated histological improvement and increases in the number and diameter of the hair follicles. EGF expression on the root sheath of hair follicles was up-regulated in both Group I and II. TGF-${\beta}$1 expression on the root sheath of hair follicles was unchanged in both Group I and II. VEGF expression in the tissues surrounding hair follicles was up-regulated in both groups. iNOS expression in the tissues surrounding hair follicles was down-regulated in both groups. Conclusions : These results suggest that Yonnyuniksoogobon-dan promotes hair growth and this effect is related to up-regulation of EGF and VEFG expression and down-regulation of TGF-${\beta}$1 and iNOS expression on hair roots.

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Effect of Kami-chungsimyeunjatang on atopic dermatitis-like skin lesions induced in NC/Nga mice by mite antigen stimulation (가미청심연자탕(加味淸心蓮子場)이 NC/Nga mice의 아토피양(樣) 피부염에 미치는 영향)

  • Han, Jae-Kyung;Kim, Yun-Hee;Yoon, Ji-Yeon
    • The Journal of Pediatrics of Korean Medicine
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    • v.21 no.1
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    • pp.87-116
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    • 2007
  • Objectives : The purpose of this study is to examine of the effect of Kami-chungsimyeunjatang(KCSYJT) medicine on the atopy eruption control. Methods : The expression of IgE, IL-4, IL-6, $TNF-{\alpha}$, IgG2b, IgM, IgG2a and IgG1 level in serum, and $IFN-{\gamma}$ production by KCSYJT were analyzed. CD3e+/CD69+, CD4+/CD25+, B220+/IgE+ and B220+/CD23+ positive cells by flow cytometry in splenocytes were assayed and the revelation of CD3e+/CD69+, CD4+/CD8+ and CD4+/CD25+ marker in PBMC, spleen and DLN were observed. The outturn of IL-4, eotaxin 2, CCR3, TARC mRNA in splenocytes werw observed. We also analyzed NC/Nga mice's ear, DLN and neck-back skin after biopy and dye by H&E, and toluidine staining (mast cells marker) method, measured about epidermis and dermis part in comparison with control group. Results : NC/Nga mice suffered from dermatitis very similar to human AD with IgE hyperproduction. Specially, result that measure IgE content in serum on 8 weeks, 12 weeks, 16 weeks decreased remarkably than control group. After experiment end, result that observe revelation CD3e+/CD69+, CD4+/CD8+ and CD4+/CD25+ marker in PBMC, spleen and DLN establishment observed recover as normal with political background. And decreased than result control group which measure IL-4, IL-6, $TNF-{\alpha}$, IgG2b, IgM, IgG2a, IgG1 level in serum, and $IFN-{\gamma}$ production secreted in Th1 cell displayed increase by KCSYJT medicines. Ear thickness decrease than control group in result that observe effect that get in ear of a NC/Nga mouse. Course inflammation immunocyte etc.. permeated of result that effect that KCSYJT medicines get to NC/Nga mouse's skin establishment analyzes ear, DLN and neck-back skin after biopy, and dye by H&E, and toluidine staining (mast cells marker) method decreased about epidermis. and inflammation of dermis part remarkably than control group. Immunohistochemical examination of the skin lesion showed decrease by KCSYJT medicines on numbers of mast cells (CCR3) and CD4+ T cells containing IL-4 necessary for IgE. Conclusions : Th1 cell and Th2 cell was observed to be shift by secretion amount of IL-4 and $IFN-{\gamma}$ by KCSYJT medicines. Therefore, the KCSYJT medicine turned out to be useful in allergy autoimmune disease.

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Functional Connections of the Vestibulo-spino-adrenal Axis in the Control of Blood Pressure Via the Vestibulosympathetic Reflex in Conscious Rats

  • Lu, Huan-Jun;Li, Mei-Han;Li, Mei-Zhi;Park, Sang Eon;Kim, Min Sun;Jin, Yuan-Zhe;Park, Byung Rim
    • The Korean Journal of Physiology and Pharmacology
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    • v.19 no.5
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    • pp.427-434
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    • 2015
  • Significant evidence supports the role of the vestibular system in the regulation of blood pressure during postural movements. In the present study, the role of the vestibulo-spino-adrenal (VSA) axis in the modulation of blood pressure via the vestibulosympathetic reflex was clarified by immunohistochemical and enzyme immunoassay methods in conscious rats with sinoaortic denervation. Expression of c-Fos protein in the intermediolateral cell column of the middle thoracic spinal regions and blood epinephrine levels were investigated, following microinjection of glutamate receptor agonists or antagonists into the medial vestibular nucleus (MVN) and/or sodium nitroprusside (SNP)-induced hypotension. Both microinjection of glutamate receptor agonists (NMDA and AMPA) into the MVN or rostral ventrolateral medullary nucleus (RVLM) and SNP-induced hypotension led to increased number of c-Fos positive neurons in the intermediolateral cell column of the middle thoracic spinal regions and increased blood epinephrine levels. Pretreatment with microinjection of glutamate receptor antagonists (MK-801 and CNQX) into the MVN or RVLM prevented the increased number of c-Fos positive neurons resulting from SNP-induced hypotension, and reversed the increased blood epinephrine levels. These results indicate that the VSA axis may be a key component of the pathway used by the vestibulosympathetic reflex to maintain blood pressure during postural movements.

Beneficial Effect of the Combination of Oral Administration and Herbal -Acupuncture Stimulation with Several Herb-combind Prescription on Streptozotocin-Induced Diabetic Rats (한약복합처방(韓藥複合處方) 약침(藥鍼) 및 경구투여(經口投與)가 Streptozotoin에 의한 흰쥐의 당뇨병(糖尿病)과 항산화능(抗酸化能)에 미치는 영향)

  • Park, Sa-hyun;Cho, Su-in;Chae, Woo-seok;Cho, Myung-rae
    • Journal of Acupuncture Research
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    • v.22 no.1
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    • pp.1-11
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    • 2005
  • Objective : The present study was carried out to investigate the preventive effect of Several Herb-combind Prescription(SHP) on Streptozotocin (STZ) -induced Diabetes mellitus. Methods : SHP was given to rats with the combination of oral administration and herbal-acupuncture stimulation. The experimental animals were divided into 3 groups : normal group of rats, control group of STZ-induced diabetic rats, sample group with SHP treatment. In vitro test of SHP showed ${\alpha}$-glucosidase inhibition, DPPH radical scavenging activity and inhibition of lipid peroxidation. Experimental diabetes was induced by the injection of STZ(60mg/kg) to the rat via the peritoneum. The effect of SHP on STZ-induced diabetes was observed by measuring the seum level of insulin, glucose, triglyceride, total cholesterol and lipid peroxides. Hepatic activities of catalase and reduced glutathione were examined and insulin granule was observed by immunohistochemical examination. Result : STZ caused hyperglycemia and hypoinsulinemia by a selectively destroying pancreatic ${\beta}$-cell. SHP treatment protected them from the hyperglycemia and hypoinsulinemia. STZ induced increase of serum triglyceride lowered by SHP treatment. And by SHP treatment, pancrease showed a big area with positive immuno-reactivity for presence of insulin with many insulin granules distributed in the ${\beta}$-cells in the islets of Langerhans. Contusions : The SHP treatment showed protective effect on diabetic rat model, and action mechanism of the effect was thought to be concerned with anti-oxidative stress.

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Study of the Expression of FasL and of Apoptosis in Gastric Epithelial Dysplasia and Gastric Adenocarcinomas (위상피이형성과 위암종에서 FasL의 발현 및 Apoptosis에 관한 연구)

  • Park Gun Uk;Han Sang Young;Lee Jong Hun;Keum Dong Joo;Roh Myung Hwan;Choi Seok Ryeol;Kim Jong Seong;Roh Mee Sook
    • Journal of Gastric Cancer
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    • v.1 no.2
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    • pp.83-91
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    • 2001
  • Purpose: This study was to observe whether the apoptotic function of tumor-infiltrating lymphocytes (TIL) is induced in human gastric epithelial dysplasia and gastric adenocarcinoma according to the role of FasL expression. Materials and Methods: A total of 56 gastric epithelial dysplasia and gastric adenocarcinoma patients were enrolled in this study: 9 cases of gastric epithelial dysplasia, 18 cases of early gastric carcinomas (EGC) and 29 cases of advanced gastric carcinomas (AGC). Immunohistochemical staining was performed for FasL and CD45, and the terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) method was used to detect cell death in tumor-infiltrating lymphocytes. Results: 1) Positive reactions of FasL to neoplastic cells were $88.9\%$ (8/9) in gastric epithelial dysplasia, $83.3\%$ (15/18) in EGC, and $75.9\%$ (22/29) in AGC. 2) Expression of TIL was decreased in the FasL positive region and was increased in the FasL negative region, and significant expression of TIL was observed in the AGC group (P=0.001). 3) Expression of apoptotic TIL was very similar to the FasL expression, and $100\%$ expression was observed in gastric epithelial dysplasia group. 4) Expression of apoptotic TIL was increased in the FasL positive region and decreased in the FasL negative region, and significant apoptotic expression was observed in the gastric epithelial dysplasia and EGC groups (P=0.0420, P=0.0263, respectively). Conclusion: These results suggest that FasL is a prevalent mediator of immune privilege in epithelial dysplasia and cancer of the stomach.

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Measurement of Mucin Amounts Using RTO3 in Patients with Chronic Airway Disease (만성 기도질환에서 흰쥐 뮤신에 대한 단 세포군 항체(RTO3)를 이용한 뮤신 측정에 관한 연구)

  • Kim, Do-Jin;Kim, Ki-Up;NamGung, Eun-Kyang;Uh, Soo-Taek;Kim, Young-Hoon;Shin, Chan-Young;Ko, Kwang-Ho;Park, Choon-Sik
    • Tuberculosis and Respiratory Diseases
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    • v.47 no.6
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    • pp.786-796
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    • 1999
  • Background: It has been anticipated that the amount and composition of mucin are changed in patients with chronic airway diseases. We evaluated whether RTO3(mAb against rat tracheal mucins) could quantify the amount of mucin from the airway in the patients with chronic airway diseases. Methods and results; 1) RTO3 was bound to high molecular weight of mucin based on Western blot in sputum and BALF from patients with chronic airway diseases. 2) The goblet cells and submucosal glands in main bronchus from human were observed by PAS stain. And immunohistochemical stain with RTO3 showed immunoreactivity on some goblet cells. 3) The amount of mucin was more increased in patients with chronic airway diseases compared to those in normal subjects. 4) In the exacerbation of asthmatics, mucin amounts were more increased than stable asthmatics. Conclusion: We suggested that secreted mucin in chronic airway diseases can be quantified by ELISA with RTO3.

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A Study on the Effects of Bee Venom Aqua-Acupuncture on Pain related Neuronal activity in the Spinal Cord (봉독약침(蜂毒藥鍼)이 척수내(脊髓內) 통증관련(痛症關聯) 신경세포(神經細胞)의 활성(活性)에 미치는 영향(影響))

  • Jeong, Sun-Hee;Lee, Jae-Dong;Koh, Hyung-Kyun;Ahn, Byoung-Choul;Choi, Do-Young;Park, Dong-Suk
    • Journal of Acupuncture Research
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    • v.17 no.2
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    • pp.153-168
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    • 2000
  • Introduction : In spite of the use of Bee Venom aqua-acupuncture in the clinics, the scientific evaluation on effects is not enough. Bee Venom aqua-acupuncture is used according to the stimulation of acupuncture point and the chemical effects of Bee Venom. The aims of this study is to investigate the analgegic effects of the Bee Venom aqua-acupuncture, through the change of writhing reflex and the change of c-fos in secondary neurons in the spinal cord. Materials and Methods : Pain animal model was used acetic acid method. The changes of writhing reflex of the mice which were derived pain by injecting acetic acid into the abdomen, after stimulating Bee Venom aqua-acupuncture on Chungwan(CV12) were measured. We used Fos immunohistochemical technique to study the neuronal activity in the spinal cord. Results : 1. Expression of c-fos in superficial dorsal horn(SDH), nucleus proprius(NP) and neck of dorsal hom(N) on 6~9th thoracic spine decreased significantly at $2.5{\times}10-4$g/kg Bee Venom aqua-acupuncture, compared with saline-acetic acid group. 2. The numeral change of Fos-LI neurons on the NP, N, and ventral gray(V) on 6-9th thoracic spine, SDH on 9-11th thoracic spine, and SDH and V on 11~13th thoracic spine decreased significantly at Chungwan(CV12) Bee Venom aqua-acupuncture, compared with saline-acetic acid group. 3. The correlation between the numbers of writhing refleax and Fos-LI neurons in T6-13 segment was statistically statistically significant at Chungwan(CV12) Bee Venom aqua-acupuncture. Conclusion : This study shows that the Bee Venom aqua-acupuncture on Chungwan(CV12) decreases the numbers of Fos-LI neurons. As the analgegic effects of Bee Venom aqua-acupuncture is recognized. Bee Venom aqua-acupuncture treatment is expected for pain modulation. In order to use it in many ways, more researches are needed for the dose and stability of Bee Venom aqua-acupuncture.

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Angiogenesis in newly regenerated bone by secretomes of human mesenchymal stem cells

  • Katagiri, Wataru;Kawai, Takamasa;Osugi, Masashi;Sugimura-Wakayama, Yukiko;Sakaguchi, Kohei;Kojima, Taku;Kobayashi, Tadaharu
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.39
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    • pp.8.1-8.8
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    • 2017
  • Background: For an effective bone graft for reconstruction of the maxillofacial region, an adequate vascular network will be required to supply blood, osteoprogenitor cells, and growth factors. We previously reported that the secretomes of bone marrow-derived mesenchymal stem cells (MSC-CM) contain numerous growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor $(TGF)-{\beta}1$, and vascular endothelial growth factor (VEGF), which can affect the cellular characteristics and behavior of regenerating bone cells. We hypothesized that angiogenesis is an important step for bone regeneration, and VEGF is one of the crucial factors in MSC-CM that would enhance its osteogenic potential. In the present study, we focused on VEGF in MSC-CM and evaluated the angiogenic and osteogenic potentials of MSC-CM for bone regeneration. Methods: Cytokines in MSC-CM were measured by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were cultured with MSC-CM or MSC-CM with anti-VEGF antibody (MSC-CM + anti-VEGF) for neutralization, and tube formation was evaluated. For the evaluation of bone and blood vessel formation with micro-computed tomography (micro-CT) and for the histological and immunohistochemical analyses, a rat calvarial bone defect model was used. Results: The concentrations of IGF-1, VEGF, and $TGF-{\beta}1$ in MSC-CM were $1515.6{\pm}211.8pg/mL$, $465.8{\pm}108.8pg/mL$, and $339.8{\pm}14.4pg/mL$, respectively. Tube formation of HUVECs, bone formation, and blood vessel formation were increased in the MSC-CM group but decreased in the MSC-CM + anti-VEGF group. Histological findings suggested that new bone formation in the entire defect was observed in the MSC-CM group although it was decreased in the MSC-CM + anti-VEGF group. Immunohistochemistry indicated that angiogenesis and migration of endogenous stem cells were much more abundant in the MSC-CM group than in the MSC-CM + anti-VEGF group. Conclusions: VEGF is considered a crucial factor in MSC-CM, and MSC-CM is proposed to be an adequate therapeutic agent for bone regeneration with angiogenesis.