골관절염에서 줄기세포를 이용한 연골 재생의 최신 지견

선종근,최익선,고지욱 대한정형외과학회 2019 대한정형외과학회지 Vol.54 No.6

Osteoarthritis is a disease characterized by the progression of articular cartilage erosion, that increases pain during joint motion and reduces the ability to withstand mechanical stress, which in turn limits joint mobility and function. Damage to articular cartilage due to trauma or degenerative injury is considered a major cause of arthritis. Numerous studies and attempts have been made to regenerate articular cartilage. In the case of partial degenerative cartilage changes, microfracture and autologous chondrocyte implantation have been proposed as surgical treatment methods, but they have disadvantages such as insufficient mutual binding to the host cells, inaccurate cell delivery, and deterioration of healthy cartilage. Stem cell-based therapies have been developed to compensate for this. This review summarizes the drawbacks and consequences of various cartilage regeneration methods and describes the various attempts to treat cartilage damage. In addition, this review will discuss cartilage regeneration, particularly mesenchymal stem cell engineering-based therapies, and explore how to treat future cartilage regeneration using mesenchymal stem cells. 골관절염은 관절연골 침식의 진행을 특징적으로 보이는 질환으로 관절운동 중에 통증을 증가시키고 기계적 스트레스를 견디는 능력을 감소시켜 결과적으로 관절의 가동성과 기능을 저하시킨다. 외상 또는 퇴행성으로 인한 관절연골의 손상이 일반적 관절염의 주요 원인으로 생각되며 이러한 관절연골 손상의 재생에 관한 수많은 연구와 시도들이 현재까지 진행되어 오고 있다. 현재까지 연골 손상의 경우 미세골절술과 자가연골세포 이식술이 일반적인 수술적 치료방법으로 제시되어 왔으나 비교적 양호한 임상 결과에도 불구하고 정상 유리연골의 생성이 미흡하여 시간이 경과하면서 결과가 악화되는 등 단점이 있다. 이를 보완하기 위하여 줄기세포 기반 치료법이 개발되었다. 이 종설에서는 현재 사용되는 다양한 연골 재생 방법들의 장단점 및 결과에 대해 요약하고 특히 중간엽 줄기세포(mesenchymal stem cells) 기반 연골 재생 치료법을 논하고 나아가 이상적인 미래 연골 재생 치료법에 대해서도 고민해보고자 한다.

The Effect of Cultured Perichondrial Cell Sheet Covered Highly Active Engineered Cartilage: in vivo Comparative Assessment

박세일,문영미,정재호,장광호,안면환 한국임상수의학회 2011 한국임상수의학회지 Vol.28 No.5

A special mesenchymal tissue layer called perichondrium has a chondrogenic capacity and is a candidate tissue for engineering of cartilage. To overcome limited potential for chondrocyte proliferation and re-absorption, we studied a method of cartilage tissue engineering comprising chondrocyte-hydrogel pluronic complex (CPC) and cultured perichondrial cell sheet (cPCs) which entirely cover CPC. For effective cartilage regeneration, cell-sheet engineering technique of high-density culture was used for fabrication of cPCs. Hydrogel pluronic as a biomimetic cell carrier used for stable and maintains the chondrocytes. The human cPCs was cultured as a single layer and entirely covered CPC. The tissue engineered constructs were implanted into the dorsal subcutaneous tissue pocket on nude mice (n = 6). CPC without cPCs were used as a controls (N = 6). Engineered cartilage specimens were harvested at 12 weeks after implantation and evaluated with gross morphology and histological examination. Biological analysis was also performed for glycosaminoglycan (GAG) and type II collagen. Indeed, we performed additional in vivo studies of cartilage regeneration using canine large fullthickness chondrial defect model. The dogs were allocated to the experimental groups as treated chondrocyte sheets with perichondrial cell sheet group (n = 4), and chondrocyte sheets only group (n = 4). The histological and biochemical studies performed 12 weeks later as same manners as nude mouse but additional immunofluorescence study. Grossly, the size of cartilage specimen of cPCs covered group was larger than that of the control. On histological examination, the specimen of cPCs covered group showed typical characteristics of cartilage tissue. The contents of GAG and type II collagen were higher in cPCs covered group than that of the control. These studies demonstrated the potential of such CPC/cPCs constructs to support chondrogenesis in vivo. In conclusion, the method of cartilage tissue engineering using cPCs supposed to be an effective method with higher cartilage tissue gain. We suggest a new method of cartilage tissue engineering using cultured perichondrial cell sheet as a promising strategy for cartilage tissue reconstruction. 조직공학적 인공연골재생에 대한 관심이 증가함에 따라 많은 연구들이 활발히 수행되고 있으나 임상적인 적용의 한계를 극복하기위한 고효능을 보유한 양질의 연골조직생산의 필요성이 증가되고 있다. 인공연골은 자연연골과는 달리 ‘연골막(perichondrium)’을 포함하고 있지 않기 때문에 장기간 생체 내에 삽입된 후에 서서히 흡수 또는 변형으로 임상적 활용에 한계가 있다고 있다. 이에 본 연구는 양질의 연골조직생산을 목적으로, 세포판 제작기법(cell sheet engineering technique) 을 기반으로 한 인체유래의 배양 연골막(cultured perichondrium)을 이용하여 만든 인공연골막세포판(cultured perichondrial cell sheet)의 생체 내 특성을 비교 분석하고, 배양된 연골막을 피복하여 고효능화를 유도한 인공연골복합체의 생체내 재생효능 및 조직특성을 비교 평가하고자 하였다. 본 연구에서는 thymic nude mouse 의 피하이식모델(study 1, n = 12)을 이용하여 담체로 hydrogel을 이용한 배양연골막 복합체의 생체내 효능을 분석하였고, 중대형동물의 대량연골 결손시의 재생효능을 평가하기 위하여 개의 무릎연골에 1 × 2 cm의 대량연골 결손모델(study 2, n = 12)을 통하여 인공배양세포판을 이식하였다. 이식12주 후 이식편을 회수하여 생화학, 분자생물학 및 면역조직학 분석을 시행한 결과, 배양연골막 복합체의 생체내 효능이 단독이식군에 비해 변형이나 과증식 없이 우수한 결과를 나타내었다. 본 연구의 결과로 토대로 배양연골막을 피복한 인공연골막의 관절내 효과를 규명하여 실제 임상적용을 조기화하는 기반을 제공하고 인공연골의 문제점이었던 변형과 흡수를 줄인 고효능 인공연골 제작기법을 제공하는데 유용할 것으로 기대된다.

Progress in biomechanical stimuli on the cell-encapsulated hydrogels for cartilage tissue regeneration

Shiva Taheri,Hanieh Sadat Ghazali,Zahra Sadat Ghazali,아미타바 바타차리아,노인섭 한국생체재료학회 2023 생체재료학회지 Vol.27 No.00

Background : Worldwide, many people suffer from knee injuries and articular cartilage damage every year, which causes pain and reduces productivity, life quality, and daily routines. Medication is currently primarily used to relieve symptoms and not to ameliorate cartilage degeneration. As the natural healing capacity of cartilage damage is limited due to a lack of vascularization, common surgical methods are used to repair cartilage tissue, but they cannot prevent massive damage followed by injury. Main body : Functional tissue engineering has recently attracted attention for the repair of cartilage damage using a combination of cells, scaffolds (constructs), biochemical factors, and biomechanical stimuli. As cyclic biomechanical loading is the key factor in maintaining the chondrocyte phenotype, many studies have evaluated the effect of biomechanical stimulation on chondrogenesis. The characteristics of hydrogels, such as their mechanical properties, water content, and cell encapsulation, make them ideal for tissue-engineered scaffolds. Induced cell signaling (biochemical and biomechanical factors) and encapsulation of cells in hydrogels as a construct are discussed for biomechanical stimulation-based tissue regeneration, and several notable studies on the effect of biomechanical stimulation on encapsulated cells within hydrogels are discussed for cartilage regeneration. Conclusion : Induction of biochemical and biomechanical signaling on the encapsulated cells in hydrogels are important factors for biomechanical stimulation-based cartilage regeneration.

연골막하 연골 결손부에 삽입한 제 1형 아교질 지지체의 연골 재생 효과

이혁구,손대구,한기환,김준형,이소영 대한성형외과학회 2005 Archives of Plastic Surgery Vol.32 No.4

The purpose of this research is to find out the degree of cartilage regeneration by inserting the atelo- collagen scaffold obtained from dermis of a calf on cartilage defect site. Dissection underneath the perichondrium by the periosteal elevator on both side of ears of six New Zealand white rabbits were made to expose the cartilage, leaving pairs of circular holes 3, 6, 9 mm width with punches. One hole was left for a control, and on the other hole atelo-collagen scaffold of the same size was transplanted. In postoperative 1, 2, 4 weeks, the tissues were dyed. The length of long axis of neocartilage was measured through an optical microscope with a 0.1mm graduation at original magnification, ×40. In the first and second week, both group showed no sign of cartilage regeneration. In the fourth week, regeneration on marginal portions was observed on all groups and the average values of length of long axis of neocartilage according to defect size were as follows: In the cases with 3mm defect, it was 0.85±0.30mm in the control group, and 1.85±0.38mm in the graft group; in the cases with 6 mm defect, 1.33±0.58mm in the control group, and 2.25±0.46mm in the graft group; and in the cases with 9mm defect, 2.33±0.77mm in the control group, and 4.47±1.39mm in the graft group. This means that the collagen scaffold has an influence on the regeneration of neocartilage. But the relative ratio of the length of neocartilage to cartilage defect size was not significant in the statistics.

Hybrid Scaffolds Composed of Hyaluronic Acid and Collagen for Cartilage Regeneration

( Hyun Jung Kim ),( Kab Keun Kim ),( Il Kyu Park ),( Baek Sun Choi ),( Jae Ho Kim ) 한국조직공학·재생의학회 2012 조직공학과 재생의학 Vol.9 No.2

Hybrid scaffolds composed of hyaluronic acid (HA) and collagen was prepared and evaluated for cartilage regeneration. The hybrid scaffolds prepared by adding 0.1, 0.3 or 0.5 wt.% collagen to HA had a three-dimensional structure with interconnected pores and showed an increase in tensile strength with increasing collagen concentration. The degradation period of the hybrid scaffolds in vitro increased with increasing collagen concentration and that the cell growth in the hybrid scaffolds increased with increasing collagen concentration for 2 weeks of cell culture. After the hybrid scaffolds with different collagen concentrations were implanted into cartilage defects of rabbit ears for 6 months, the GAG concentration of the hybrid scaffolds was higher than the HA scaffold itself, suggesting that cartilage tissue was easily formed in the collagen-containing scaffolds although the GAG concentration in the hybrid scaffolds decreased with increasing collagen concentration. Therefore, it is concluded that the collagen- containing porous scaffolds can be effectively used for cartilage regeneration.

Preconditioning of Rabbit Mesenchymal Stem Cells in Polyglycolic Acid (PGA) Scaffold using Low-Intensity Ultrasound Improved Regeneration of Cartilage in Rabbit Articular Cartilage Defect Model

( Ji Hao Cui ),( So Ra Park ),( Byung Hyune Choi ),( Byoung Hyun Min ) 한국조직공학·재생의학회 2010 조직공학과 재생의학 Vol.7 No.1

This study investigated the effect of low intensity ultrasound (LIUS) stimulation of mesenchymal stem cells (MSCs) in vitro on the repair of cartilage defect after implantation of the construct in vivo. Rabbit MSCs were cultured in the polyglycolic acid (PGA) scaffold and preconditioned with (MSCs/US+) or without (MSCs/US-) LIUS stimulation during the chondrogenic differentiation for 1 week in vitro. The LIUS stimulation was carried out at the intensity of 200 mW/Cm2 every day for 20 min over a week. The constructs were implanted into the cartilage defects created in the rabbit femoral trochlea. The defect only was used as a negative control and rabbit chondrocytes seeded in PGA was used as a positive control, respectively. The repair of cartilage defect was examined at 2, 4, 8 and 12 weeks after implantation, respectively. The gross observation showed that the articular cartilage defects were filled with the repaired tissue in all groups. Histological and immunohistochemical analyses revealed, however, more intensive and widespread expression of proteoglycans and type II collagen in the MSCs/US+ group than in the MSCs/US- group. Fibrous tissues were observed mainly in the defect only group. The chondrocytes groups showed efficient repair of the defect by hyaline cartilage. In conclusion, this study suggested that LIUS preconditioning of MSCs in vitro could be an effective method to promote chondrogenesis of MSCs and repair of cartilage defect in vivo.

Tissue Adhesive Hydrogel for Stem Cell Transplantation for Bone and Cartilage Regeneration

조정호,이정승,신지수,권성근,조승우 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.1

Catechol-modified hyaluronic acid (HA-CA) hydrogel has shown highly adhesive and biocompatible properties, which are beneficial in cell transplantation. Here, we synthesized HA-CA hydrogel and evaluated its efficacy in stem cell transplantation, especially for bone and cartilage regeneration. When human adipose-derived stem cells (hADSCs) were encapsulated with bone morphogenetic protein 2 (BMP-2) in the hydrogel, osteogenic differentiation of hADSCs was greatly improved, and ultimately, in vivo bone regeneration was significantly facilitated in a critical-sized calvarial defect mouse model. In addition, adhesive property of HA-CA hydrogel allowed for efficient and stable retention of cells on the decellularized trachea matrix, which would enhance cartilage regeneration in vivo. ^**This study was supported by a grant (2016R1A5A1004694) from Translational Research Center for Protein Function Control (TRCP) funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea.

High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model

Gun Hee Cho,Bae Hyun Cheol,조원영,정의만,Hee Jung Park,Ha Ru Yang,Sun Young Wang,You Jung Kim,Shin Dong-Myung,Hyung-Min Chung,In Gyu Kim,한혁수 한국생체재료학회 2023 생체재료학회지 Vol.27 No.00

Background Mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo. Methods Flow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites. Results FreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs’ function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O’Driscoll scores showing that more hyaline cartilage was formed on the defects. Conclusion FreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy.

A Combinational Therapy of Articular Cartilage Defects: Rapid and Effective Regeneration by Using Low-Intensity Focused Ultrasound After Adipose Tissue-Derived Stem Cell Transplantation

Song Byeong-Wook,Park Jun-Hee,Kim Bomi,Lee Seahyoung,Lim Soyeon,Kim Sang Woo,Choi Jung-Won,Lee Jiyun,Kang Misun,Hwang Ki-Chul,Chae Dong-Sik,Kim Il-Kwon 한국조직공학과 재생의학회 2020 조직공학과 재생의학 Vol.17 No.3

BACKGROUND: Although low-intensity pulsed ultrasound has been reported to be potential cartilage regeneration, there still unresolved treatment due to cartilage fibrosis and degeneration by a lack of rapid and high-efficiency treatment. The purpose of this study was to investigate the effect of a combination therapy of focused acoustic force and stem cells at site for fast and efficient healing on cartilage regeneration. METHODS: Using a rat articular cartilage defects model, one million adipose tissue-derived stem cells (ASCs) were injected into the defect site, and low-intensity focused ultrasound (LOFUS) in the range of 100–600 mV was used for 20 min/day for 2 weeks. All experimental groups were sacrificed after 4 weeks in total. The gross appearance score and hematoxylin and eosin (H&E), Alcian blue, and Safranin O staining were used for measuring the chondrogenic potential. The cartilage characteristics were observed, and type II collagen, Sox 9, aggrecan, and type X collagen were stained with immunofluorescence. The results of the comprehensive analysis were calculated using the Mankin scoring method. RESULTS: The gross appearance scores of regenerated cartilage and chondrocyte-like cells in H&E images were higher in LOFUS-treated groups compared to those in negative control or ASC-treated groups. Safranin O and Alcian blue staining demonstrated that the 100 and 300 mV LOFUS groups showed greater synthesis of glycosaminoglycan and proteoglycan. The ASC ? LOFUS 300 mV group showed positive regulation of type II collagen, Sox 9 and aggrecan and negative regulation of type X collagen, which indicated the occurrence of cartilage regeneration based on the Mankin score result. CONCLUSION: The combination therapy, which involved treatment with ASC and 300 mV LOFUS, quickly and effectively reduced articular cartilage defects.

Delivery of growth factor-associated genes to mesenchymal stem cells for cartilage and bone tissue regeneration

Ahn, Jongchan,Park, Seah,Cha, Byung-Hyun,Kim, Jae Hwan,Park, Hansoo,Joung, Yoon Ki,Han, Inbo,Lee, Soo-Hong Techno-Press 2014 Biomaterials and biomedical engineering Vol.1 No.3

Genetically-modified mesenchymal stem cells (GM-MSCs) have emerged as promising therapeutic tools for orthopedic degenerative diseases. GM-MSCs have been widely reported that they are able to increase bone and cartilage tissue regeneration not only by secreting transgene products such as growth factors in a long-term manner, also by inducing MSCs into tissue-specific cells. For example, MSCs modified with BMP-2 gene increased secretion of BMP-2 protein resulting in enhancement of bone regeneration, while MSCs with TGF-b gene did cartilage regeneration. In this review, we introduce several growth factors for gene delivery to MSCs and strategies for bone and cartilage tissue regeneration using GM-MSCs. Furthermore, we describe strategies for strengthening GM-MSCs to more intensively induce tissue regeneration by co-delivery system of multiple genes.