• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권6호
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• pp.511-519
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• 2006

Autogenous bone grafts have been considered the gold standard for maxillofacial bony defects. However, this procedure could entail a complicated surgical procedure as well as potential donor site morbidity. Possibly the best solution for bone-defect regeneration is a tissue engineering approach, i.e. the use of a combination of a suitable scaffold with osteogenic cells. A major source of osteogenic cells is the bone marrow. Bone marrow-derived mesenchymal stem cells are multipotent and have the ability to differentiate into osteoblastic, chondrocytic, and adipocytic lineage cells. However, the isolation of cells from bone marrow has someproblems when used in clinical setting. Bone marrow aspiration is sometimes potentially more invasive and painful procedure and carries of a risk of morbidity and infection. A minimally invasive, easily accessible alternative would be cells derived from periosteum. The periosteum also contains multipotent cells that have the potential to differentiate into osteoblasts and chondrocytes. In the present study, we evaluated the osteogenic activity and mineralization of cultured human periosteal-derived cells. Periosteal explants were harvested from mandibule during surgical extraction of lower impacted third molar. The periosteal cells were cultured in the osteogenic inductive medium consisting of DMEM supplemented with 10% fetal calf serum, 50g/ml L-ascorbic acid 2-phosphate, 10 nmol dexamethasone and 10 mM -glycerophosphate for 42 days. Periosteal-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 14 of culture period, then decreased in intensity during the culture period. ALP mRNA expression increased up to day 14 with a decrease thereafter. Osteocalcin mRNA expression appeared at day 7 in culture, after that its expression continuously increased in a time-dependent manner up to the entire duration of culture. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. In conclusion, our study showed that cultured human periosteal-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix. As the periosteal-derived cells, easily harvested from intraoral procedure such as surgical extraction of impacted third molar, has the excellent potential of osteogenic capacity, tissue-engineered bone using periosteal-derived cells could be the best choice in reconstruction of maxillofacial bony defects.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제29권3호
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• pp.197-205
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• 2007

Angiogenesis is a essential part for bone formation and bone fracture healing. Vascular endothelial growth factor (VEGF), one of the most important molecules among many angiogenic factors, is a specific mitogen for vascular endothelial cells. VEGF-mediated angiogenesis is required for bone formation and repair. However, the effect of VEGF on osteoblastic cells during osteogenesis is still controversial. In recent days, substantial progress have been made toward developing tissue-engineered alternatives to autologous bone grafting for maxillofacial bony defects. Periosteum has received considerable interest as a better source of adult stem cells. Periosteum has the advantage of easy harvest and contains various cell types and progenitor cells that are able to differentiate into a several mesenchymal lineages, including bone. Several studies have reported the bone formation potential of periosteal cells, however, the correlation between VEGF signaling and cultured human periosteal cell-derived osteogenesis has not been fully investigated yet. The purpose of this study was to examine the correlation between VEGF signaling and cultured human periosteal-derived cells osteogenesis. Periosteal tissues of $5\;{\times}\;20\;mm$ were obtained from mandible during surgical extraction of lower impacted third molar from 3 patients. Periosteal-derived cells were introduced into the cell culture and were subcultured once they reached confluence. After passage 3, the periosteal-derived cells were further cultured for 42 days in an osteogenic inductive culture medium containing dexamethasone, ascorbic acid, and ${\beta}-glycerophosphate$. We evaluated the alkaline phosphatase (ALP) activity, the expression of Runx2 and VEGF, alizarin red S staining, and the quantification of osteocalcin and VEGF secretion in the periosteal-derived cells. The ALP activity increased rapidly up to day 14, followed by decrease in activity to day 35. Runx2 was expressed strongly at day 7, followed by decreased expression at day 14, and its expression was not observed thereafter. Both VEGF 165 and VEGF 121 were expressed strongly at day 35 and 42 of culture, particularly during the later stages of differentiation. Alizarin red S-positive nodules were first observed on day 14 and then increased in number during the entire culture period. Osteocalcin and VEGF were first detected in the culture medium on day 14, and their levels increased thereafter in a time-dependent manner. These results suggest that VEGF secretion from cultured human periosteal-derived cells increases along with mineralization process of the extracellular matrix. The level of VEGF secretion from periosteal-derived cells might depend on the extent of osteoblastic differentiation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권5호
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• pp.341-345
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• 2010

Introduction: Skeletal homeostasis is normally maintained by the stability between bone formation by osteoblasts and bone resorption by osteoclasts. However, the correlation between the inflammatory reaction and osteoblastic differentiation of cultured osteoprogenitor cells has not been fully investigated. This study examined the effects of inflammatory cytokines on the osteoblastic differentiation of cultured human periosteal-derived cells. Materials and Methods: Periosteal-derived cells were obtained from the mandibular periosteum and introduced into the cell culture. After passage 3, the periosteal-derived cells were further cultured in an osteogenic induction Dulbecco's modified Eagle's medium (DMEM) medium containing dexamethasone, ascorbic acid, and $\beta$-glycerophosphate. In this culture medium, tumor necrosis factor (TNF)-$\alpha$ with different concentrations (0.1, 1, and 10 ng/mL) or interleukin (IL)-$1{\beta}$ with different concentrations (0.01, 0.1, and 1 ng/mL) were added. Results: Both TNF-$\alpha$ and IL-$1{\beta}$ stimulated alkaline phosphatase (ALP) expression in the periosteal-derived cells. TNF-$\alpha$ and IL-$1{\beta}$ increased the level of ALP expression in a dose-dependent manner. Both TNF-$\alpha$ and IL-$1{\beta}$ also increased the level of alizarin red S staining in a dose-dependent manner during osteoblastic differentiation of cultured human periosteal-derived cells. Conclusion: These results suggest that inflammatory cytokines TNF-$\alpha$ and IL-$1{\beta}$ can stimulate the osteoblastic activity of cultured human periosteal-derived cells.

• 생명과학회지
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• 제28권12호
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• pp.1455-1460
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• 2018

최근 들어 급속한 고령화 사회가 진행되고 있으며 이로 인해 골관절염과 골다공증 등의 퇴행성 골질환 환자수도 동반하여 증가하고 있다. 따라서 고령화에 따른 골관련 질환의 새로운 제어와 치료법 개발을 위해 성체줄기세포의 골세포 분화유도를 활용한 재생의학도 활발히 연구되고 있다. 또한 관련 연구에서 줄기세포의 분화과정에서 미토콘드리아의 산화적인산화가 중요하다고 알려지고 있다. 흥미롭게도 최근 연구에서 아스피린의 주성분인 salicylate가 동물세포의 미토콘드리아 생합성을 증진시키는 효과가 보고되었다. 그러나 성체줄기세포에서 salicylate가 골세포분화나 미토콘드리아 생합성을 유도할 수 있는지에 대한 연구결과는 미비한 실정이다. 본 연구에서는 인체 골막 유래의 성체줄기세포를 이용하여 골세포분화나 미토콘드리아 생합성에 대한 salicylate의 영향을 분석하였다. 골막 유래 성체줄기세포의 골세포 분화유도 과정에 동반한 salicylate 처리는 잘 알려진 골세포분화 표지자인 alkaline phosphatase의 활성을 증가시키는 결과를 본 연구에서 얻었다. 이러한 연구결과는 salicylate가 줄기세포로부터 골세포로의 분화를 조절할 수 있는 물질이 될 수 있음을 제시한다. 또한 이러한 골세포 분화과정에서 미토콘드리아 생합성도 salicylate 처리에 의해 증가됨이 관찰되었다. 따라서, 미토콘드리아 생합성이나 기능을 조절하는 물질이 성체줄기세포의 골세포 분화과정에도 영향을 줄 수 있으며, 이러한 물질이 골세포분화나 재생의학의 새로운 조절 물질로 응용될 수 있음을 제시한다.

• KSBB Journal
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• 제20권3호
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• pp.233-237
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• 2005

누에체액 성분 중의 30K 단백질은 Q-Sepharose FF를 이용한 단계별 농도구배 이온교환 크로마토그래피로 0.16 M NaCl 용출 조건에서 쉽게 얻을 수 있었다. 얻어진 단백질을 인간유래 정상세포에 처리하여 세포 증식 향상이 확인되었고, 이는 30K 단백질이 인간을 위한 세포치료제에 이용되는 세포의 증식을 위한 첨가물로 사용될 수 있음으로써 의료 분야에의 적용 가능성을 확인하였다. 또한 본 연구를 통해 확립된 30K 단백질의 간단한 정제 공정은 향후 대량 생산 공정으로 scale-up이 용이하게 이루어질 것으로 기대된다. 이 결과들은 동물세포를 생산 숙주로 사용할 때 생산 물질 자체의 생산량 증대에도 효과적으로 이용될 수 있으며 세포치료제를 위한 세포의 증식에도 효과를 보일 수 있으므로 세포배양을 이용한 생산 공정 전반에 큰 영향을 미칠 수 있을 것이다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제31권5호
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• pp.440-453
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• 2005

Objectives : To develop a bioactive membrane for guided bone regeneration (GBR), the biocompatibility and bone regenerating capacity of the cellulose membrane obtained from the Ascidians squirt skin were evaluated. Materials and methods : After processing the pure cellulose membrane from the squirt skin, the morphological study, amino acid analysis and the immunoreactivity of the cellulose membrane were tested. Total eighteen male Spraque-Dawley rats (12 weeks, weighing 250 to 300g) were divided into two control (n=8) and another two experimental groups (n=10). In the first experimental group (n=5), the cellulose membrane was applicated to the 8.0 mm sized calvarial bone defect and the same sized defect was left without cellulose membrane in the first control group (n=4). In the another experimental group (n=5), the cellulose membrane was applicated to the same sized calvarial bone defect after femoral bone graft and the same sized defect with bone graft was left without cellulose membrane in the another control group (n=4). Each group was sacrificed after 6 weeks, the histological study with H&E and Masson trichrome stain was done, and immunohistochemical stainings of angiogenin and VEGF were also carried out. Results : The squirt skin cellulose showed the bio-inductive effect on the bone and mesenchymal tissues in the periosteum of rat calvarial bone. This phenomenon was found only in the inner surface of the cellulose membrane after 6 weeks contrast to the outer surface. Bone defect covered with the bioactive cellulose membrane showed significantly greater bone formation compared with control groups. Mesenchymal cells beneath the inner surface of the bioactive cellulose membrane were positive to the angiogenin and VEGF antibodies. Conclusion : We suppose that there still remains extremely little amount of peptide fragment derived from the basement membrane matrix proteins of squirt skin, which is a kind of anchoring protein composed of glycocalyx. This composition could prevent the adverse immunological hypersensitivity and also induce bioactive properties of cellulose membrane. These properties induced the effective angiogenesis with rapid osteogenesis beneath the inner surface of cellulose membrane, and so the possibilities of clinical application in dental field as a GBR material will be able to be suggested.

• 생명과학회지
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• 제29권12호
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• pp.1337-1344
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• 2019

골다공증의 진행은 뼈질량 감소와 골절위험 증가를 야기한다. 골다공증은 노인 인구에서 흔하며, 최근 들어 급속한 고령화 사회로 인해 그 환자수도 동반하여 크게 증가하고 있다. 현재 처방되는 골다공증 치료제의 대부분은 파골세포 억제 효과에 기반하여 골흡수를 방지한다. 그러나 이러한 골다공증 치료제는 새로운 뼈형성을 증가시키지는 못하며 수반되는 여러 부작용도 보고되고 있다. 따라서 골다공증의 새로운 제어와 치료법 개발을 위해 성체줄기세포의 골세포 분화유도와 조골세포 활성을 도모하는 재생의학적 접근이 활발히 연구되고 있다. 스타틴(statin) 계열 약물은 혈중 콜레스테롤 강하제로 심혈관 질환에 흔히 처방되는 치료제이다. 흥미롭게도 최근 일련의 연구에서 이러한 스타틴이 조골세포 활성에 긍정적인 영향을 주어 뼈형성을 촉진한다는 보고가 발표되고 있다. 따라서, 본 연구에서는 이러한 스타틴 약물이 인체 골막유래 성체줄기세포의 골세포 분화과정이나 조골세포 활성에 영향이 있는 지를 분석하였다. 현재 임상적으로 처방되는 총 7 종류의 스타틴 약물에 대해, 골막유래 성체줄기세포의 골세포 분화과정에서 조골세포 활성과 관련된 초기와 후기 표지자인 alkaline phosphatase의 활성과 칼슘 침착을 각각 분석하였다. 본 연구에서 일부 스타틴(pitavastatin과 pravastatin)은 약하지만 뼈형성을 증가시키는 효과가 있음을 알 수 있었다. 이러한 연구결과는 스타틴이 골막유래 줄기세포로부터 골세포로의 분화나 조골세포 활성을 조절할 수 있는 물질이 될 수 있으며, 이러한 약물이 골세포분화나 재생의학의 새로운 조절 물질로서 골다공증 치료에 응용될 수 있음을 제시한다.