Bone Tissue Engineering Using Marrow Stromal Cells

Jo, In-Ho,Lee, Jung-Min,Suh, Hwal,Kim, Hyong-Bum Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.1

Bone tissue defects cause a significant socioeconomic problem, and bone is the most frequently transplanted tissue beside blood. Autografting is considered the gold standard treatment for bone defects, but its utility is limited due to donor site morbidity. Hence, much research has focused on bone tissue engineering as a promising alternative method for repair of bone defects. Marrow stromal cells (MSCs) are considered to be potential cell sources for bone tissue engineering. In bone tissue engineering using MSCs, bone is formed through intramembranous and endochondral ossification in response to osteogenic inducers. Angiogenesis is a complex process mediated by multiple growth factors and is crucial for bone regeneration. Vascular endothelial growth factor plays important roles in bone tissue regeneration by promoting the migration and differentiation of osteoblasts, and by inducing angiogenesis. Scaffold materials used for bone tissue engineering include natural components of bone, such as calcium phosphate and collagen I, and biodegradable polymers such as poly(lactide-coglycolide). However, ideal scaffolds for bone tissue engineering have yet to be found. Bone tissue engineering has been successfully used to treat bone defects in several human clinical trials to regenerate bone defects. Through investigation of MSC biology and the development of novel scaffolds, we will be able to develop advanced bone tissue engineering techniques in the future.

PLLA/HA Composite Scaffold와 골수 줄기세포를 이용한 조직공학적 골재생에 대한 연구

김병렬,장현석,임재석,이의석,김동현,Kim, Byeong-Yol,Jang, Hyon-Seok,Rim, Jae-Suk,Lee, Eui-Seok,Kim, Dong-Hyun 대한악안면성형재건외과학회 2008 Maxillofacial Plastic Reconstructive Surgery Vol.30 No.4

Aim of the study: Scaffolds are crucial to tissue engineering/regeneration. Biodegradable polymer/ceramic composite scaffolds can overcome the limitations of conventional ceramic bone substitutes such as brittleness and difficulty in shaping. In this study, poly(L-lactide)/hydroxyapatite(PLLA/HA) composite scaffolds were fabricated for in vivo bone tissue engineering. Material & methods: In this study, PLLA/HA composite microspheres were prepared by double emulsion-solvent evaporation method, and were evaluated in vivo bone tissue engineering. Bone marrow mesenchymal stem cell from rat iliac crest was differentiated to osteoblast by adding osteogenic medium, and was mixed with PLLA/HA composite scaffold in fibrin gel and was injected immediately into rat cranial bone critical size defect(CSD:8mm in diameter). At 1. 2, 4, 8 weeks after implantation, histological analysis by H-E staining, histomorphometric analysis and radiolographic analysis were done. Results: BMP-2 loaded PLLA/HA composite scaffolds in fibrin gel delivered with osteoblasts differentiated from bone marrow mesenchymal stem cells showed rapid and much more bone regeneration in rat cranial bone defects than control group. Conclusion: This results suggest the feasibility and usefulness of this type of scaffold in bone tissue engineering.

Effect of TNF-α and IL-6 on Compact Bone-Derived Cells

Zhang Yiming,Li Xianqi,Chihara Takahiro,Dong Hongwei,Kagami Hideaki 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.3

Background: Although bone tissue engineering has already been applied clinically, its regeneration efficacy is not always sufficient. Local inflammatory cytokines are considered as the major factors that induce apoptosis of transplanted cells, thus leading to insufficient new bone formation. In this study, we focused on the effects of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) on differentiation and apoptosis of compact bone-derived cells (CBDCs). Methods: CBDCs were obtained from mouse legs and cultured. The effects of TNF-α and/or IL-6 on the osteogenic differentiation and apoptosis of CBDCs were analyzed in vitro. To confirm the expression of local inflammatory cytokines in vivo, CBDCs were transplanted to the back of immunocompetent mice. Results: IL-6 exerted inconsistent effects on the expression of the different osteogenic markers tested, while significantly upregulating Fas. By contrast, the addition of TNF-α dramatically reduced the expression of all tested osteogenic markers and increased Fas expression. The highest dose of IL-6 could partially reverse the repressive effect of TNF-α, while the addition of IL-6 further increased Fas expression in CBDCs compared to TNF-α alone. The results from in vivo experiments showed the presence of transplants with and without new bone formation. The transplants without bone formation were characterized by higher IL-6 and lower IL-10 expression than those with bone formation, while the expression of TNF-α did not show notable difference. Conclusion: The results of this study suggest an important role for IL-6 in modulating the efficacy of bone tissue engineering, which can affect osteogenic cells both positively and negatively. Background: Although bone tissue engineering has already been applied clinically, its regeneration efficacy is not always sufficient. Local inflammatory cytokines are considered as the major factors that induce apoptosis of transplanted cells, thus leading to insufficient new bone formation. In this study, we focused on the effects of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) on differentiation and apoptosis of compact bone-derived cells (CBDCs). Methods: CBDCs were obtained from mouse legs and cultured. The effects of TNF-α and/or IL-6 on the osteogenic differentiation and apoptosis of CBDCs were analyzed in vitro. To confirm the expression of local inflammatory cytokines in vivo, CBDCs were transplanted to the back of immunocompetent mice. Results: IL-6 exerted inconsistent effects on the expression of the different osteogenic markers tested, while significantly upregulating Fas. By contrast, the addition of TNF-α dramatically reduced the expression of all tested osteogenic markers and increased Fas expression. The highest dose of IL-6 could partially reverse the repressive effect of TNF-α, while the addition of IL-6 further increased Fas expression in CBDCs compared to TNF-α alone. The results from in vivo experiments showed the presence of transplants with and without new bone formation. The transplants without bone formation were characterized by higher IL-6 and lower IL-10 expression than those with bone formation, while the expression of TNF-α did not show notable difference. Conclusion: The results of this study suggest an important role for IL-6 in modulating the efficacy of bone tissue engineering, which can affect osteogenic cells both positively and negatively.

Effects of Induction Culture on Osteogenesis of Scaffold-Free Engineered Tissue for Bone Regeneration Applications

박혜민,김선화,최병현,Sang-Hyug Park 한국조직공학과 재생의학회 2022 조직공학과 재생의학 Vol.19 No.2

BACKGROUND: Restoration of the bone defects caused by infection or disease remains a challenge in orthopedic surgery. In recent studies, scaffold-free engineered tissue with a self-secreted extracellular matrix has been proposed as an alternative strategy for tissue regeneration and reconstruction. Our study aimed to engineer and fabricate self-assembled osteogenic and scaffold-free tissue for bone regeneration. METHODS: Osteogenic scaffold-free tissue was engineered and fabricated using fetal cartilage-derived progenitor cells, which are capable of osteogenic differentiation. They were cultured in osteogenic induction environments or using demineralized bone powder for differentiation. The fabricated tissue was subjected to real-time qPCR, biochemical, and histological analyses to estimate the degree of in vitro osteogenic differentiation. To demonstrate bone formation in an in vivo environment, scaffold-free tissue was transplanted into the dorsal subcutaneous site of nude mice. Bone development was monitored postoperatively over 8 weeks by the observation of calcium deposition in the matrix. RESULTS: In the in vitro experiments, engineered osteogenically induced scaffold-free tissue demonstrated three-dimensional morphological characteristics, and sufficient osteogenic differentiation was confirmed through the quantification of specific osteogenic gene markers expressed and calcium accumulation within the matrix. Following the evaluation of differentiation efficacy, in vivo experiments revealed distinct bone formation, and that blood vessels had penetrated the fabricated tissue. CONCLUSION: The novel engineering of scaffold-free tissue with osteogenic potential can be used as an optimal bone graft substitute for bone regeneration.

Decellularized Human Adipose Tissue as an Alternative Graft Material for Bone Regeneration

Ahn Woo Beom,Lee Yu Bin,Ji Yi-Hwa,Moon Kyoung-Sik,Jang Hyon-Seok,Kang Sun-Woong 한국조직공학과 재생의학회 2022 조직공학과 재생의학 Vol.19 No.5

BACKGROUND: Tissue engineering approaches to treat damaged bone include various tissue transplants such as autologous, allogeneic, and xenografts. Artificial materials have been widely introduced to meet the demand for graft materials, but insufficiency in supply is still not resolved. In this study, human adipose tissue, easily obtained from the human body, was harvested, and the tissue was decellularized to fabricate a decellularized human adipose tissue matrix (DM) as an alternative graft material. METHODS: Human adipose tissue was obtained via liposuction. The obtained fresh adipose tissue sample was cut into pieces then put into decellularization solution (1% antibiotic–antimycotic solution and 1% phenylmethanesulphonyl fluoride). Lipids were further removed via treatment in isopropanol. The sample was then subjected to another enzymatic digestion and lipid removal processes. The obtained decellularized adipose tissue matrix was lyophilized to form a graft material in disc shape. RESULTS: Decellularization was confirmed by nuclear staining methods and detection of RNA and DNA via PCR. Bone morphogenetic protein 2 (BMP2)-loaded DM showed the ability to form new bone tissue when implanted in subcutaneous tissue. In recovery of a mouse calvarial defect model, BMP2-loaded DM exhibited similar levels of bone tissue regeneration efficiency compared with a well-defined commercial product, BMP2-loaded CollaCote. CONCLUSION: The DM developed in this study is expected to address the problem of insufficient supply of graft materials and contribute to the treatment of bone defects of critical size as an alternative bone graft material with preserved extracellular matrix components.

Review: Scaffold Characteristics, Fabrication Methods, and Biomaterials for the Bone Tissue Engineering

Jae-Won Jang,Kyung-Eun Min,Cheolhee Kim,Jesik Shin,Jiwoon Lee,Sung Yi 한국정밀공학회 2023 International Journal of Precision Engineering and Vol.24 No.3

The goal of tissue engineering is to replace or regenerate damaged tissue. Scaffold fabrications and biomaterial selections are crucial factors for artificial tissue and bone tissue engineering, which are important due to the limited availability of tissue donors. This paper reviews the scaffold design considerations, manufacturing methods, and biomaterials for bone tissue engineering, and discusses current challenges and future perspectives. Scaffolds are required to have non-hazardous properties such as biocompatibility and biodegradability for the human body, and the necessary mechanical properties to support body weight, or to perform other roles, depending on the type of tissue. Moreover, scaffold structures such as porosity, pore size, and pore shape should be optimized to achieve cell viability and proliferation. Many conventional fabrication methods including thermally induced phase separation, emulsion freeze-drying, solvent casting, gas forming, and electrospinning have been studied and developed, but 3D printing is more suitable for bone tissue engineering because of its ability to manufacture complicated structures. Biomaterials can be divided into four categories: polymer, ceramic, metal, and composites. Composites blend two or more biomaterials to achieve desired properties for matching individual patient conditions. Finding a balance between fabrication method and biomaterial selection, in order to match properties between the scaffold and the target tissue, will be key to the field of bone tissue engineering in the future.

Tissue-Engineered Hydroxyapatite Bone Scaffold Impregnated with Osteoprogenitor Cells Promotes Bone Regeneration in Sheep Model

Bajuri Mohd Yazid,Selvanathan Nanchappan,Dzeidee Schaff Fatin Nadira,Abdul Suki Muhammad Haziq,Ng Angela Min Hwei 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.3

BACKGROUND: Managing massive bone defects, a great challenge to orthopaedics reconstructive surgery. The problem arise is the supply of suitable bone is limited with many complications. Tissue-engineered hydroxyapatite bone (TEHB) scaffold impregnated with osteoprogenitor cells developed as an alternative to promote bone regeneration. METHODS: This animal protocol has been approved by Universiti Kebangsaan Malaysia Animal Ethical Committee. The TEHB scaffold prepared from hydroxyapatite using gel casting method. A total of six adolescent female sheep were chosen for this study. Later, all the sheep were euthanized in a proper manner and the bone harvested for biomechanical study. Bone marrow was collected from iliac crest of the sheep and bone marrow stem cells (BMSCs) isolated and cultured. BMSCs then cultured in osteogenic medium for osteoprogenitor cells development and the plasma collected was seeded with osteoprogenitor cells mixed with calcium chloride. Bone defect of 3 cm length of tibia bone created from each sheep leg and implanted with autologous and TEHB scaffold in 2 different groups of sheep. Wound site was monitored weekly until the wound completely healed and conventional X-ray performed at week 1 and 24. Shear test was conducted to determine the shear force on the autologous bone and TEHB scaffold after implantation for 24 weeks. RESULTS: All of the sheep survived without any complications during the study period and radiograph showed new bone formation. Later, the bone harvested was for biomechanical study. The highest shear force for the autologous group was 13 MPa and the lowest was 5 MPa while for the scaffold group, the highest was 10 MPa and the lowest was 3 MPa. Although, proximal and distal interface of autologous bone graft shows higher shear strength compared to the TEHB scaffold but there is no significant difference in both groups, p value > 0.05. Histologically in both proximal and distal interface in both arms shows bone healing and woven bone formation. CONCLUSION: TEHB scaffold impregnated with osteoprogenitor cells has the potential to be developed as a bone substitute in view of its strength and capability to promote bone regeneration. BACKGROUND: Managing massive bone defects, a great challenge to orthopaedics reconstructive surgery. The problem arise is the supply of suitable bone is limited with many complications. Tissue-engineered hydroxyapatite bone (TEHB) scaffold impregnated with osteoprogenitor cells developed as an alternative to promote bone regeneration. METHODS: This animal protocol has been approved by Universiti Kebangsaan Malaysia Animal Ethical Committee. The TEHB scaffold prepared from hydroxyapatite using gel casting method. A total of six adolescent female sheep were chosen for this study. Later, all the sheep were euthanized in a proper manner and the bone harvested for biomechanical study. Bone marrow was collected from iliac crest of the sheep and bone marrow stem cells (BMSCs) isolated and cultured. BMSCs then cultured in osteogenic medium for osteoprogenitor cells development and the plasma collected was seeded with osteoprogenitor cells mixed with calcium chloride. Bone defect of 3 cm length of tibia bone created from each sheep leg and implanted with autologous and TEHB scaffold in 2 different groups of sheep. Wound site was monitored weekly until the wound completely healed and conventional X-ray performed at week 1 and 24. Shear test was conducted to determine the shear force on the autologous bone and TEHB scaffold after implantation for 24 weeks. RESULTS: All of the sheep survived without any complications during the study period and radiograph showed new bone formation. Later, the bone harvested was for biomechanical study. The highest shear force for the autologous group was 13 MPa and the lowest was 5 MPa while for the scaffold group, the highest was 10 MPa and the lowest was 3 MPa. Although, proximal and distal interface of autologous bone graft shows higher shear strength compared to the TEHB scaffold but there is no significant difference in both groups, p value > 0.05. Histologically in both proximal and distal interface in both arms shows bone healing and woven bone formation. CONCLUSION: TEHB scaffold impregnated with osteoprogenitor cells has the potential to be developed as a bone substitute in view of its strength and capability to promote bone regeneration.

HA/TCP 골이식재상에 이식된 지방유래 줄기세포의 골모세포로의 분화 및 골형성에 대한 연구

임재석,권종진,장현석,이의석,정유민,이태형,박정균,Rim, Jae-Suk,Gwon, Jong-Jin,Jang, Hyon-Seok,Lee, Eui-Seok,Jeong, You-Min,Lee, Tai-Hyung,Park, Jeong-Kyun 대한악안면성형재건외과학회 2010 Maxillofacial Plastic Reconstructive Surgery Vol.32 No.2

Aim of the study: An alternative source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Adipose tissue could be processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). This study was performed to confirm the availability of ATSCs in bone tissue engineering. Materials amp; Methods: In this study, adipose tissue-derived mesenchymal stem cell was extracted from the liposuctioned abdominal fat of 24-old human and cultivated, and the stem cell surface markers of CD 105 and SCF-R were confirmed by immunofluorescent staining. The proliferation of bone marrow mesenchymal stem cell and ATSCs were compared, and evaluated the osteogenic differentiation of ATSCs in a specific osteogenic induction medium. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining. Expression of osteocyte specific BMP-2, ALP, Cbfa-1, Osteopontin and osteocalcin were confirmed by RT-PCR. With differentiation of ATSCs, calcium concentration was assayed, and osteocalcin was evaluated by ELISA (Enzyme-linked immunosorbant assay). The bone formation by 5-week implantation of HA/TCP block loaded with bone marrow mesenchymal stem cells and ATSCs in the subcutaneous pocket of nude mouse was evaluated by histologic analysis. Results: ATSCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes was also detected. ATSCs could be easily identified through fluorescence microscopy, and bone formation in vivo was confirmed by using ATSC-loaded HA/TCP scaffold. Conclusions: The present results show that ATSCs have an ability to differentiate into osteoblasts and formed bone in vitro and in vivo. So ATSCs may be an ideal source for further experiments on stem cell biology and bone tissue engineering.

Microengineered Platforms for Co-Cultured Mesenchymal Stem Cells towards Vascularized Bone Tissue Engineering

박혜련,임동진,성민희,이수홍,나도균,박한수 한국조직공학과 재생의학회 2016 조직공학과 재생의학 Vol.13 No.5

Bone defects are common disease requiring thorough treatments since the bone is a complex vascularized tissue that is composed of multiple cell types embedded within an intricate extracellular matrix (ECM). For past decades, tissue engineering using cells, proteins, and scaffolds has been suggested as one of the promising approaches for effective bone regeneration. Recently, many researchers have been interested in designing effective platform for tissue regeneration by orchestrating factors involved in microenvironment around tissues. Among factors affecting bone formation, vascularization during bone development and after minor insults via endochondral and intramembranous ossification is especially critical for the long-term support for functional bone. In order to create vascularized bone constructs, the interactions between human mesenchymal stem cells (MSCs) and endothelial cells (ECs) have been investigated using both direct and indirect co-culture studies. Recently, various culture methods including micropatterning techniques, three dimensional scaffolds, and microfluidics have been developed to create micro-engineered platforms that mimic the nature of vascularized bone formation, leading to the creation of functional bone structures. This review focuses on MSCs co-cultured with endothelial cells and microengineered platforms to determine the underlying interplay between co-cultured MSCs and vascularized bone constructs, which is ultimately necessary for adequate regeneration of bone defects.

Artificial Bone via Bone Tissue Engineering: Current Scenario and Challenges

Shivaji Kashte,Amit Kumar Jaiswal,Sachin Kadam 한국조직공학과 재생의학회 2017 조직공학과 재생의학 Vol.14 No.1

Bone provides mechanical support, and flexibility to the body as a structural frame work along with mineral storage, homeostasis, and blood pH regulation. The repair and/or replacement of injured or defective bone with healthy bone or bone substitute is a critical problem in orthopedic treatment. Recent advances in tissue engineering have shown promising results in developing bone material capable of substituting the conventional autogenic or allogenic bone transplants. In the present review, we have discussed natural and synthetic scaffold materials such as metal and metal alloys, ceramics, polymers, etc. which are widely being used along with their cellular counterparts such as stem cells in bone tissue engineering with their pros and cons.