Bone regeneration in a rabbit ulna defect model: use of allogeneic adipose-derivedstem cells with low immunogenicity

Tissue engineering provides new potential treatments for the repair of bone defects. Bone-marrow-derived mesenchymal stem cells (BMSCs) represent an attractive cell source for therapeutic applications involving tissue engineering, although disadvantages, such as pain of harvest and low proliferation efficiency, are major limitations to the application of BMSCs in the clinic. Adipose-derived stem cells (ASCs) with their multilineage potential and satisfactory proliferation potential can be induced into the osteogenic lineage in vitro and can be anchored onto suitable scaffolds as seed cells to repair bone defects successfully in an autologous setting. Previous studies have indicated that both undifferentiated BMSCs and ASCs exhibit immunosuppression and immunoprivilege properties. We compare the immuno-function of undifferentiated and osteo-differentiated ASCs in vitro and explore the feasibility of applying allogeneic ASCs to the repair of ulnar bone defects in the rabbit model. Our study demonstrates that allogeneic osteogenic differentiated ASCs maintain low immunogenicity and negative immunomodulation. The allogeneic osteogenic differentiated ASCs combined with demineralized bone matrix successfully regenerate ulnar bone defects in rabbits without immunosuppressive therapies.     

Regeneration of articular cartilage by adipose tissue derived mesenchymal stem cells: Perspectives from stem cell biology and molecular medicine

Adipose‐derived stem cells (ASCs) have been discovered for more than a decade. Due to the large numbers of cells that can be harvested with relatively little donor morbidity, they are considered to be an attractive alternative to bone marrow derived mesenchymal stem cells. Consequently, isolation and differentiation of ASCs draw great attention in the research of tissue engineering and regenerative medicine. Cartilage defects cause big therapeutic problems because of their low self‐repair capacity. Application of ASCs in cartilage regeneration gives hope to treat cartilage defects with autologous stem cells. In recent years, a lot of studies have been performed to test the possibility of using ASCs to re‐construct damaged cartilage tissue. In this article, we have reviewed the most up‐to‐date articles utilizing ASCs for cartilage regeneration in basic and translational research. Our topic covers differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes, increased cartilage formation by co‐culture of ASCs with chondrocytes and enhancing chondrogenic differentiation of ASCs by gene manipulation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

小型猪ASCs生物学性状研究及其骨组织工程应用探讨

目的:观察小型猪脂肪来源干细胞(adipose derived stem/stromal cells,ASCs)的形态学特点,研究其多向分化潜能和生物学特性,探讨其在骨组织工程方面的应用。方法:切取雄性小型猪背部皮下脂肪,体外分离培养并鉴定ASCs,观察其生长、增殖特性和组织学形态;并诱导其向脂肪细胞和骨细胞多向分化,分别采用油红O染色和茜素红染色鉴定,细胞表面分子通过流式细胞术鉴定。结果:贴壁的ASCs为长梭形,生长增殖快,性状稳定,流式细胞术检测显示CD29、CD90阳性表达,CD14、CD31、CD34阴性表达。通过相应的诱导培养液诱导,ASCs可向脂肪细胞和骨细胞分化并表现出相应的生物学特征。结论:小型猪背部皮下脂肪取材方便,可获得大量脂肪用以分离ASCs,ASCs增殖分化能力较强,在条件培养基诱导下可成骨分化,可作为种子细胞应用于骨组织工程。     

Activation and promotion of adipose stem cells by tumour necrosis factor‐alpha preconditioning for bone regeneration

There is a major medical need for developing novel and effective approaches for repairing non‐union and critical‐sized bone defects. Although the mechanisms remain to be determined, it is known that inflammation plays a crucial role in initiating bone repair and regeneration. This study investigated the effect of short‐term (3 days) preconditioning with tumor necrosis factor‐alpha (TNF‐α) on proliferation, mobilization, and differentiation of adipose tissue‐derived mesenchymal stem cells (ASCs). We demonstrated that TNF‐α pre‐conditioning increased proliferation, mobilization, and osteogenic differentiation of ASCs and up‐regulated bone morphogenetic protein‐2 (BMP‐2) protein level. BMP‐2 silencing by siRNA partially inhibited osteogenic differentiation of ASCs induced by TNF‐α; BMP‐2 pre‐conditioning also significantly increased osteogenic differentiation of ASCs but the effects were significantly smaller than those observed for TNF‐α preconditioning. Furthermore, TNF‐α treatment promoted extracellular‐signal‐regulated kinases(Erk)1/2 and p38 mitogen‐activated protein kinase (MAPK) signaling pathways, but only Erk1/2 inhibition reduced the BMP‐2 levels and osteogenic differentiation induced by TNF‐α preconditioning. Together, these results support the hypothesis that inflammation contributes to bone regeneration by promoting proliferation, mobilization, and osteogenic differentiation of ASCs; 3 days of TNF‐α preconditioning, mimicking the short boost of inflammation normally occurring after bone injury, might serve as a feasible approach for directing stem cells into osteogenic differentiation. J. Cell. Physiol. 9999: XX–XX, 2013. © 2013 Wiley Periodicals, Inc. J. Cell. Physiol. 228: 1737–1744, 2013. © 2013 Wiley Periodicals, Inc.     

Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold

Recent studies have shown that liposuction aspirates from rat, rabbit, mouse, and human sources contain pluripotent adipose tissue-derived stromal cells (ASCs) that can differentiate into various mesodermal cell types, including osteoblasts, myoblasts, chondroblasts, and preadipocytes. To develop a research model for autologous bone tissue engineering, we isolated ASCs from human liposuction aspirates (hASCs) and induced their osteogenic differentiation in three-dimensional poly(dl-lactic-co-glycolic acid) (PLGA) scaffolds. Human liposuction aspirates were proteolytically digested and centrifuged to obtain hASCs. After primary culture in control media and expansion to three passages, the cells were seeded in two-dimensional plates or three-dimensional PLGA scaffolds and cultured in osteogenic media for 4 weeks. In two-dimensional culture, osteogenesis was assessed by RT-PCR analysis of the osteogenic-specific bone sialoprotein mRNA, by alkaline phosphatase staining, and by von Kossa staining. In three-dimensional culture, osteogenesis was assessed by von Kossa and alizarine red S staining at 1, 2, and 4 weeks following osteogenic induction. hASCs incubated in two-dimensional osteogenic media stained positively for alkaline phosphatase and with von Kossa stain after 2 weeks of differentiation. Expression of the osteogenesis-specific bone sialoprotein gene was detected by RT-PCR after 2 weeks of differentiation. PLGA scaffolds seeded with hASCs showed multiple calcified extracellular matrix nodules by von Kossa and alizarine red S staining after 2 weeks of differentiation. In conclusion, the authors identified an osteogenic potential of hASCs and demonstrated osteogenic differentiation of hASCs into an osteogenic lineage in three-dimensional PLGA scaffolds.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cellsAdipose mesenchymal stem cells in the field of bone tissue engineering

Bone tissue engineering represents one of the most challenging emergent fields for scientists and clinicians.Current failures of autografts and allografts in many pathological conditions have prompted researchers to find new biomaterials able to promote bone repair or regeneration with specific characteristics of biocompatibility,biodegradability and osteoinductivity.Recent advancements for tissue regeneration in bone defects have occurred by following the diamond concept and combining the use of growth factors and mesenchymal stem cells(MSCs).In particular,a more abundant and easily accessible source of MSCs was recently discovered in adipose tissue.These adipose stem cells(ASCs)can be obtained in large quantities with little donor site morbidity or patient discomfort,in contrast to the invasive and painful isolation of bone marrow MSCs.The osteogenic potential of ASCs on scaffolds has been examined in cell cultures and animal models,with only a few cases reporting the use of ASCs for successful reconstruction or accelerated healing of defects of the skull and jaw in patients.Although these reports extend our limited knowledge concerning the use of ASCs for osseous tissue repair and regeneration,the lack of standardization in applied techniques makes the comparison between studies difficult.Additional clinical trials are needed to assess ASC therapy and address potential ethical and safety concerns,which must be resolved to permit application in regenerative medicine.     

骨质疏松大鼠脂肪基质细胞成骨能力的研究

目的:探讨骨质疏松大鼠的脂肪基质细胞有无成骨能力。方法:采用去势法构建SD大鼠骨质疏松模型,设置2组样本,分别为19月龄骨质疏松SD雌性大鼠,正常19月龄SD雌性大鼠。每组大鼠分别取腹股沟脂肪垫中脂肪,用胶原酶消化法培养获得脂肪间充质干细胞,绘制并分析细胞生长曲线;再用经典的骨向诱导液诱导2组ASCs,比较2组细胞骨向分化能力有无差别。结果:骨质疏松大鼠与正常老龄大鼠的ASCs骨向分化能力无明显差别。结论:可用去势法构建SD大鼠骨质疏松模型,并可取其细胞行进一步研究。老龄骨质疏松大鼠的ASCs也可以考虑作为种子细胞促进其自体骨愈合,从而为利用自体ASCs促进骨质疏松骨愈合打下一定的理论基础。     

Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation in vitro

In the den, hibernating brown bears do not develop tissue atrophy or organ damage, despite almost no physical activity. Mesenchymal stem cells could play an important role in tissue repair and regeneration in brown bears. Our objective was to determine if adipose tissue-derived stem cells (ASCs) can be recovered from wild Scandinavian brown bears and characterize their differentiation potential. Following immobilization of wild brown bears 7-10 days after leaving the den in mid-April, adipose tissue biopsies were obtained. ASCs were recovered from 6 bears, and shown to be able to undergo adipogenesis and osteogenesis in monolayer cultures and chondrogenesis in pellet cultures. Remarkably, when grown in standard cell culture medium in monolayer cultures, ASCs from yearlings spontaneously formed bone-like nodules surrounded by cartilaginous deposits, suggesting differentiation into osteogenic and chondrogenic lineages. This ability appears to be lost gradually with age. This is the first study to demonstrate stem cell recovery and growth from brown bears, and it is the first report of ASCs spontaneously forming extracellular matrix characteristic of bone and cartilage in the absence of specific inducers. These findings could have implications for the use of hibernating brown bears as a model to study disuse osteoporosis.     

脂肪干细胞在再生医学中的应用

再生医学是一门研究如何促进创伤与组织再生及功能重建的新兴学科,主要通过研究干细胞分化、机体等正常组织创伤修复与再生等机制来维持、修复、再生或改善损伤组织和器官功能。脂肪干细胞（adipose-derived stem cells,ASCs）是近年来从脂肪组织中分离得到的一种具有多向分化潜能的干细胞,是一种足量的、可用于实际的、有一定吸引力的自体细胞代替的供体资源,并能够广泛的用于组织修复、再生、发育的可塑性及细胞治疗等研究中。阐述了脂肪干细胞在旁分泌、软组织重建及损伤修复、骨骼肌重建、心血管重建、神经系统重建及癌症转移与入侵方面的作用模式,概括总结了目前利用脂肪干细胞参与的临床治疗方法,以期对脂肪干细胞在再生医学中应用研究提供参考。     

Adipose-derived stem cells: An appropriate selection for osteogenic differentiation

Mesenchymal stem cells (MSCs) are a major component of various forms of tissue engineering. MSCs have self-renewal and multidifferential potential. Osteogenic differentiation of MSCs is an area of attention in bone regeneration. One form of MSCs are adipose-derived stem cells (ASCs), which can be simply harvested and differentiated into several cell lineages, such as chondrocytes, adipocytes, or osteoblasts. Due to special properties, ASCs are frequently used in vitro and in vivo bone regeneration. Identifying factors involved in osteogenic differentiation of ASCs is important for better understanding the mechanism of osteogenic differentiation. Different methods are used to stimulate osteogenesis of ASCs in literature, including common osteogenic media, growth factors, hormones, hypoxia, mechanical and chemical stimuli, genetic modification, and nanotechnology. This review article provides an overview describing the isolation procedure, characterization, properties, current methods for osteogenic differentiation of ASCs, and their basic biological mechanism.     

MIP-3α 联合成骨诱导因子诱导大鼠脂肪干细胞向成牙本质样细胞分化*

目的:观察巨噬细胞炎性蛋白-3α(MIP-3α)对大鼠脂肪干细胞(Adipose derived stem cells,ASCs)向成牙本质样细胞体外分化作用的影响。方法:分离、培养并鉴定大鼠ASCs;以MIP-3α联合成骨诱导因子(地塞米松,β-甘油磷酸钠,以及抗坏血酸)诱导第3代大鼠ASCs向成牙本质样细胞定向分化。诱导培养1、4、7d后,分别测定碱性磷酸酶(alkaline phosphatese,ALP)活性,并用RT-PCR及Western Blot检测成牙本质细胞的标志基因dspp及标志物牙本质涎蛋白(DSP)。结果:与单独加入成骨诱导因子相比,MIP-3α与成骨诱导因子联合应用能使ALP活性、dspp的mRNA表达以及DSP升高。结论:本研究显示MIP-3α与成骨诱导因子联合应用可以增强成牙本质细胞相关基因以及蛋白的表达,为牙齿再生种子细胞的寻找开辟了一条新思路。     

Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.     

Adipose-derived stem cells alleviate osteoporosis by enchancing osteogenesis and inhibiting adipogenesis in a rabbit model

Background aimsOsteoporosis (OP) is characterized by a reduction in bone quality, which is associated with inadequacies in bone marrow mesenchymal stromal cells (BMSCs). As an alternative cell source to BMSCs, adipose-derived stem cells (ASCs) have been investigated for bone repair because of their osteogenic potential and self-renewal capability. Nevertheless, whether autologous ASCs can be used to promote bone regeneration under osteoporotic conditions has not been elucidated.MethodsThe OP rabbit model was established by means of bilateral ovariectomy (OVX). Both BMSCs and ASCs were harvested from OVX rabbits and expanded in vitro. The effects of osteogenic-induced ASCs on the in vitro adipogenic and osteogenic capabilities of BMSCs were evaluated. Autologous ASCs were then encapsulated by calcium alginate gel and transplanted into the distal femurs of OVX rabbits (n = 12). Hydrogel without loading cells was injected into the contralateral femurs as a control. Animals were killed for investigation at 12 weeks after transplantation.ResultsOsteogenic-induced ASCs were able to promote osteogenesis and inhibit adipogenesis of osteoporotic BMSCs through activation of the bone morphogenetic protein 2/bone morphogenetic protein receptor type IB signal pathway. Local bone mineral density began to increase at 8 weeks after ASC transplantation (P < 0.05). At 12 weeks, micro–computed tomography and histological evaluation revealed more new bone formation in the cell-treated femurs than in the control group (P < 0.05).ConclusionsThis study demonstrated that ASCs could stimulate proliferation and osteogenic differentiation of BMSCs in vitro and enhance bone regeneration in vivo, which suggests that autologous osteogenic-induced ASCs might be useful to alleviate OP temporally.     

Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice

Recent studies suggest that human adipose tissue contains pluripotent stem cells similar to bone marrow-derived stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. In the present study, we extend this approach to characterize adipose tissue-derived stromal cells, sometimes called processed lipoaspirate (PLA) cells. Adipose-derived stromal cells (ASCs) were isolated from inguinal fat pads of GFP transgenic mice after extensive washing with phosphate-buffered saline and treatment with collagenase. After primary culture in a control medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum) and expansion to two passages, the cells were incubated in either an osteogenic medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum+dexamethasone+ascorbate-2-phosphate+beta-glycerophosphate) or a chondrogenic medium (Dulbecco's modified Eagle's medium+1% fetal bovine serum+insulin+ascorbate-2-phosphate+transforming growth factor-beta1) for 2-4 weeks to induce osteogenesis and chondrogenesis, respectively. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining, while chondrogenic differentiation was assessed by Alcian blue staining. Expression of osteocyte specific osteopontin, osteocalcin, and alkaline phosphatase, and chondrocyte specific aggrecan and type II/X collagen was confirmed by RT-PCR. ASCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes, except osteocalcin, was also detected. Incubation with chondrogenic medium induced Alcian blue positive cells and expression of aggrecan and type II/X collagen genes. No osteochondrogenic differentiation was observed in cells incubated in the control medium. ASCs from GFP transgenic mice have both osteogenic and chondrogenic potential in vitro. Since this cell population can be easily identified through fluorescence microscopy, it may be an ideal source of ASCs for further experiments on stem cell biology and tissue engineering.     

Induction of Osteogenic Differentiation of Adipose Derived Stem Cells by Microstructured Nitinol Actuator-Mediated Mechanical Stress

The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.     

Adhesion,Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.     

0Adipose-derived stem cells: Implications in tissue regeneration

Adipose-derived stem cells(ASCs) are mesenchymal stem cells(MSCs) that are obtained from abundant adipose tissue, adherent on plastic culture flasks, can be expanded in vitro, and have the capacity to differ-entiate into multiple cell lineages. Unlike bone marrow-derived MSCs, ASCs can be obtained from abundant adipose tissue by a minimally invasive procedure, which results in a high number of cells. Therefore, ASCs are promising for regenerating tissues and organs dam-aged by injury and diseases. This article reviews the implications of ASCs in tissue regeneration.     

Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles

Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.