In-vitro- und In-vivo-Knochenregenerierung durch mesenchymale Stammzellen aus dem Nabelschnurblut

BACKGROUND: Mesenchymal stem cells with an osteoblastic differentiating potency are investigated in regard of probable tissue engineering for further clinical application. The following report describes the use of cord blood derived stem cells as an alternative to other stem cell populations for bone regenerating tissue engineering. METHODS: To demonstrate the multipotency of cord blood derived mesenchymal stem cells, unrestringated somatic stem cells (USSC) were isolated from cord blood and underwent an osteo-, chondro- and adipoblastic in vitro stimulation. To evaluate the osteoinductive potency of a porcine collagen I/III cell carrier USSC were incubated on this matrix. To investigate the in vivo effects of human USSC an athymic rat model was developed. These cells were transplanted into a femoral defect. RESULTS: Cord blood derived mesenchymal stem cells (USSC) have an in vitro multipotency and show adipo-, chondro- and osteogenic differentiation. The porcine collagen I/III carrier promoted an osteoblastic differentiation. USSC survived after xenotransplantation in an athymic rat and differentiated into osteoblasts filling the bony defect zone. CONCLUSION: Human USSC are a mesenchymal multipotent stem cell population that shows osteoblastic differentiation onto a collagen I/III carrier in vitro as well as in an athymic rat in vivo.     

Designer Stem Cells: Genome Engineering and the Next Generation of Cell‐Based Therapies

Stem cells provide tremendous promise for the development of new therapeutic approaches for musculoskeletal conditions. In addition to their multipotency, certain types of stem cells exhibit immunomodulatory effects that can mitigate inflammation and enhance tissue repair. However, the translation of stem cell therapies to clinical practice has proven difficult due to challenges in intradonor and interdonor variability, engraftment, variability in recipient microenvironment and patient indications, and limited therapeutic biological activity. In this regard, the success of stem cell‐based therapies may benefit from cellular engineering approaches to enhance factors such as purification, homing and cell survival, trophic effects, or immunomodulatory signaling. By combining recent advances in gene editing, synthetic biology, and tissue engineering, the potential exists to create new classes of “designer” cells that have prescribed cell‐surface molecules and receptors as well as synthetic gene circuits that provide for autoregulated drug delivery or enhanced tissue repair. Published by Wiley Periodicals, Inc. J Orthop Res 37:1287–1293, 2019.     

Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages

OBJECTIVES: Marrow stromal cells are mesenchymal stem cells able to differentiate into cardiomyocytes in vitro. We tested the hypothesis that marrow stromal cells, when implanted into myocardium, can undergo milieu-dependent differentiation and express cardiomyogenic phenotypes in vivo. METHODS: Isogenic adult rats were used as donors and recipients to simulate autologous transplantation. Marrow stromal cells isolated from donor leg bones were culture-expanded, labeled with 4;,6-diamidino-2-phenylindole, and then injected into the myocardium of the recipients. The hearts were harvested from 4 days to 12 weeks after implantation, and the implant sites were examined to identify the phenotypes of the labeled marrow stromal cells. RESULTS: Viable cells labeled with 4;, 6-diamidino-2-phenylindole can be identified in host myocardium at all time points after implantation. Implanted marrow stromal cells show the growth potential in a myocardial environment. After 4 weeks, donor cells derived from marrow stromal cells demonstrate myogenic differentiation with the expression of sarcomeric myosin heavy chain and organized contractile proteins. Positive staining for connexin 43 indicates the formation of gap junctions, which suggests that cells derived from marrow stromal cells, as well as native cardiomyocytes, are connected by intercalated disks. CONCLUSIONS: Different cell sources have been used as donor cells for cellular cardiomyoplasty. Our findings indicate that marrow stromal cells can also be used as donor cells. In an appropriate microenvironment they will exhibit cardiomyogenic phenotypes and may replace native cardiomyocytes lost by necrosis or apoptosis. Because marrow stromal cells can be obtained repeatedly by bone marrow aspiration and expanded vastly in vitro before being implanted or used as autologous implants, and because their use does not call for immunosuppression, the clinical use of marrow stromal cells for cellular cardiomyoplasty appears to be most advantageous.     

Prepare cells to repair the heart: mesenchymal stem cells for the treatment of heart failure

Heart failure is one of the most important cardiovascular diseases, with high mortality, and invasive treatment such as mechanical circulatory support and cardiac transplantation is sometimes required for severe heart failure. Therefore, the development of less invasive and more effective therapeutic strategies is desired. Cell therapy is attracting growing interest as a new approach for the treatment of heart failure. As a cell source, various kinds of stem/progenitor cells such as bone marrow cells, endothelial progenitor cells, mesenchymal stem cells (MSC) and cardiac stem cells have been investigated for their efficacy and safety. Especially, bone marrow-derived MSC possess multipotency and can be easily expanded in culture, and are thus an attractive therapeutic tool for heart failure. Recent studies have revealed the underlying mechanisms of MSC in cardiac repair: MSC not only differentiate into specific cell types such as cardiomyocytes and vascular endothelial cells, but also secrete a variety of paracrine angiogenic and cytoprotective factors. It has also been suggested that endogenous MSC as well as exogenously transplanted MSC migrate and participate in cardiac repair. Based on these findings, several clinical trials have just been started to evaluate the safety and efficacy of MSC for the treatment of heart failure.     

HEMOXCell,a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate‐Supplemented Media

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three‐dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)‐supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%‐increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two‐fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and “stemness.” These features will be further tested within three‐dimensional scaffolds.     

Novel Supplier of Mesenchymal Stem Cell: Subacromial Bursa

Mesenchymal stem cells (MSCs) are multipotent stromal elements that can differentiate into a variety of cell types. MSCs are good sources of therapeutic cells for degenerative diseases. For these reason, many researchers have focused on searching for other sources of MSCs. To obtain MSCs for clinical use requires surgery of the donor that therefore can induce donor morbidity, since the common sources at present are bone marrow and adipose tissues. In this study, we investigated the existence of MSCs in postoperative discarded tissues. Subacromial bursal tissues were obtained from the shoulders of 3 injured patients. The cells from the bursa tissues were isolated through treatment with collagenase. The isolated cells were then seeded and expanded by serial passaging under normal culture system. To evaluate MSC characteristics of the cells, their MSC markers were confirmed by mRNA and protein expression. Multipotent ability was assessed using differentiation media and immunohistochemistry. Cells from the bursa expressed MSCs markers—CD29, CD73, CD90, and PDGFRB (platelet-derived growth factor receptor-beta). Moreover, as to their multipotency, bursal cells differentiated into adipocytes (fat cells), osteocytes (bone cells), and chondrocytes (cartilage cells). In summary, we showed that MSCs could be generated from the subacromial bursa, which is medical waste after surgery.     

Differential hematopoietic supportive potential and gene expression of stroma cell lines from midgestation mouse placenta and adult bone marrow

During mouse embryogenesis, hematopoietic development takes place in several distinct anatomic locations. The microenvironment of different hematopoietic organs plays an important role in the proliferation and maturation of the hematopoietic cells. We hypothesized that fetal stromal cells would be distinct to adult bone marrow (BM)-derived stromal cells because the BM contributes mainly to the homeostasis of hematopoietic stem cells (HSCs), while extensive expansion of HSCs occurs during fetal development. Here we report the establishment of stromal cell lines from fetal hematopoietic organs, namely aorta-gonad-mesonephros (AGM), midgestation placenta (PL), and fetal liver (FL) together with adult bone marrow (BM). The growth patterns and hematopoietic supportive potential were studied. Their phenotypic and molecular gene expression profiles were also determined. Stromal cell lines from each tissue were able to support cobblestone area formation of BM c-Kit(+)Sca-1(+) hematopoietic cells: 22 (22/47) from AGM, three (3/4) from PL, three (3/4) from FL, and three (3/3) from BM. There were similar levels of expansion of total mononuclear cells (TMNs) when HSCs were cocultured with fetal stroma and adult BM stroma. However, PL-derived stromal cells supported higher levels of generation of colony-forming progenitor cell (CFU-C), indicated by more colonies and colonies with significantly larger size. Flow cytometric analysis of the PL1 cells demonstrated a phenotype of CD45(-), CD105(+), Sca-1(+), CD34(+), and CD49d(+), compared to adult BM1 cells, which were CD45(-), CD105(+), Sca-1(+), CD34(-), and CD49d(-). Using Affymetrix microarray analysis, we identified that genes specifically express in endothelial cells, such as Tie1, Tek, Kdr, Flt4, Emcn, Pecam1, Icam2, Cdh5, Esam1, Prom1, Cd34, and Sele were highly expressed in stroma PL1, consistent with an endothelial phenotype, while BM1 expressed a mesenchymal stromal phenotype. In summary, these data demonstrate distinct characteristics of stromal cells that provide insights into the microenvironmental control of HSCs.     

Prostatic stromal cells derived from benign prostatic hyperplasia specimens possess stem cell like property

INTRODUCTION: The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. METHODS: Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. RESULTS: Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. CONCLUSIONS: Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia.     

State of the art of where we are at using stem cells for stress urinary incontinence

AIMS: This review aims to discuss: 1) the neurophysiology, highlighting the importance of the middle urethra, and treatment of stress urinary incontinence (SUI); 2) current injectable cell sources for minimally-invasive treatment; and 3) the potential of muscle-derived stem cells (MDSCs) for the delivery of neurotrophic factors. METHODS: A PUB-MED search was conducted using combinations of heading terms: urinary incontinence, urethral sphincter, stem cells, muscle, adipose, neurotrophins. In addition, we will update the recent work from our laboratory. RESULTS: In anatomical and functional studies of human and animal urethra, the middle urethra containing rhabdosphincter, is critical for maintaining continence. Cell-based therapies are most often associated with the use of autologous multipotent stem cells, such as the bone marrow stromal cells. However, harvesting bone marrow stromal stem cells is difficult, painful, and may yield low numbers of stem cells upon processing. In contrast, alternative autologous adult stem cells such as MDSCs and adipose-derived stem cells can be easily obtained in large quantities and with minimal discomfort. Not all cellular therapies are the same, as demonstrated by the differences in safety and efficacy from muscle-sourced MDSCs versus myoblasts versus fibroblasts. CONCLUSIONS: Transplanted stem cells may have the ability to undergo self-renewal and multipotent differentiation, leading to sphincter regeneration. In addition, such cells may release, or be engineered to release, neurotrophins with subsequent paracrine recruitment of endogenous host cells to concomitantly promote a regenerative response of nerve-integrated muscle. The dawn of a new paradigm in the treatment of SUI may be near.     

Regenerative potential of mesodermal stem cells for the heart

There has been increasing interest in recent years in the phenomenon of "regeneration," especially in the function of the bone marrow stromal cell system in the support of hematopoiesis. The stromal cell system has been proposed to consist of mesodermal stem cells that are capable of self-renewal and differentiation into a variety of mesodermal tissues, including bone, cartilage, tendon, fat, endothelium, skeletal muscle, and cardiomyocytes. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of cardiomyocytes in patients with severe cardiac failure due to loss of muscle cells. Some studies have indicated that locally or systemically delivered mesodermal stem cells can generate de novo cardiomyocytes. Despite their potential clinical utility for cellular and gene therapy, the mechanism of differentiation in mesodermal stem cells and characterization of stem cells in terms of surface antigen expression remain to be resolved. Although some clinical trials have been initiated using crude bone marrow-derived stromal cells, we need more knowledge of stem cells to establish a standard protocol for cellular therapy.     

不同胚龄人神经干细胞的分离和培养

目的 探索不同胚龄人神经干细胞分离和培养的适宜条件。方法 在多种培养条件下，采用无血清培养和单细胞克隆技术，从7周和13周人胚脑中分离和培养神经干细胞，应用免疫荧光染色予以鉴定。结果 从两个不同胚龄的人胚脑中分离出具有自我更新和多分化潜能的神经干细胞，未包被组7周的人胚脑除高密度县浮培养法生长不良，其它密度及培养法均有克隆球形成；13周的人胚脑仅中等密度悬浮培养法及高密度贴壁培养法有克隆球形成；包被组各种密度培养均生长不良。结论 人胚脑中存在具有自我更新能力和多分化潜能的神经干细胞，随着胚龄增大，培养难度逐渐加大，高密度贴壁培养法适宜各胚龄神经干细胞的分离培养。     

Subpopulations of marrow stromal cells share a variety of osteoblastic markers

Summary A series of stromal cell lines derived from mouse bone marrow, representing subpopulations of putative stromal cell types, were examined for the expression of osteoblastic properties. The effects of dexamethasone and specific inhibitors on alkaline phosphatase activity, cAMP response to bone-seeking hormones, and the ability to mineralize extracellular matrixin vitro as well as collagen typing were used as osteoblastic markers. We found that all stromal cell types examined posses some osteoblastic features but differ in the degree of expression. The data provide support to the hypothesis of a common stem cell for marrow stromal cells.     

The characterization of epithelial and stromal subsets of candidate stem/progenitor cells in the human adult prostate

OBJECTIVES: Questions regarding the cell source and mechanisms in the initiation and progression of prostate cancer are today still open for debate. Indeed, our knowledge regarding prostate cell regulation, self-renewal, and cytodifferentiation is presently rather limited. In this study, we investigated these processes in the normal adult human prostate. METHODS: Dynamic expression patterns in prostate stem/progenitor cells, intermediate/transit-amplifying cells, and cell lineages were immunohistochemically identified in an in situ explant renewal model of the human normal/benign adult prostate (n=6). RESULTS: Cells with a basal phenotype proliferated significantly in explant cultures, whereas luminal cells went into apoptosis. Results further show down-regulation in tissue cultures of the basal and hypothetical stem cell marker Bcl-2 in the majority of cells, except in rare putative epithelial stem cells. Investigation of established (AC133) and novel candidate prostate stem/progenitor markers, including the cell surface receptor tyrosine kinase KIT and its ligand stem cell factor (SCF), showed that these rare epithelial cells are AC133(+)/CD133(low)/Bcl-2(high)/cytokeratin(+)/vimentin(-)/KIT(low)/SCF(low). In addition, we report on a stromal population that expresses the mesenchymal marker vimentin and that is AC133(-)/CD133(high)/Bcl-2(-)/cytokeratin(-)/KIT(high)/SCF(high). CONCLUSIONS: We provide evidence for epithelial renewal in response to tissue culture and for basal and epithelial stem/progenitor cell recruitment leading to an expansion of an intermediate luminal precursor phenotype. Data further suggest that SCF regulates prostate epithelial stem/progenitor cells in an autocrine manner and that all or a subset of the identified novel stromal phenotype represents prostate stromal progenitor cells or interstitial pacemaker cells or both.     

The promise of stem cell therapy to restore urethral sphincter function

The promise of stem cell therapy for the treatment of stress urinary incontinence is that transplanted stem cells may undergo self-renewal and potential multipotent differentiation, leading to urethral sphincter regeneration. Cell-based therapies are most often associated with the use of autologous multipotent stem cells, such as bone marrow cells. However, harvesting bone marrow stromal stem cells is difficult, painful, and may yield low numbers of stem cells. Alternatively, autologous adult stem cells, such as muscle-derived stem cells, can be obtained in large quantities and with minimal discomfort. Not all cells and cellular therapies are the same, however, and proper placement of cells into target structures may be critical to eventual treatment success. In particular, restoration and repair of the damaged urethral sphincter is crucial to maintain urinary continence because active urethral closure is largely mediated by pudendal nerves that innervate the striated muscles and rhabdosphincter of the middle urethra.     

Living scaffolds: surgical repair using scaffolds seeded with human adipose-derived stem cells

Background

Decellularized porcine small intestinal submucosa (SIS) is a biological scaffold used surgically for tissue repair. Here, we demonstrate a model of SIS as a scaffold for human adipose-derived stem cells (ASCs) in vitro and apply it in vivo in a rat ventral hernia repair model.

Study design

ASCs adherence was examined by confocal microscopy and proliferation rate was measured by growth curves. Multipotency of ASCs seeded onto SIS was tested using adipogenic, chondrogenic, and osteogenic induction media. For in vivo testing, midline abdominal musculofascial and peritoneal defects were created in Sprague–Dawley rats. Samples were evaluated for tensile strength, histopathology and immunohistochemistry.

Results

All test groups showed cell adherence and proliferation on SIS. Fibronectin-treated scaffolds retained more cells than those treated with vehicle alone (p < 0.05). Fresh stromal vascular fraction (SVF) pellets containing ASCs were injected onto the SIS scaffold and showed similar results to cultured ASCs. Maintenance of multipotency on SIS was confirmed by lineage-specific markers and dyes. Histopathology revealed neovascularization and cell influx to ASC-seeded SIS samples following animal implantation. ASC-seeded SIS appeared to offer a stronger repair than plain SIS, but these results were not statistically significant. Immunohistochemistry showed continued presence of cells of human origin in ASC-seeded repairs at 1 month postoperation.

Conclusion

Pretreatment of the scaffold with fibronectin offers a method to increase cell adhesion and delivery. ASCs maintain their immunophenotype and ability to differentiate while on SIS. Seeding freshly isolated SVF onto the scaffold demonstrated that minimally manipulated cells may be useful for perioperative surgical applications within the OR suite. We have shown that this model for a “living mesh” can be successfully used in abdominal wall reconstruction.

     

Human embryonic stem cell-derived CD34+ cells function as MSC progenitor cells

Mesenchymal stem/stromal cells (MSCs) have been isolated from various tissues and utilized for an expanding number of therapies. The developmental pathways involved in producing MSCs and the phenotypic precursor/progenitor cells that give rise to human MSCs remain poorly defined. Human embryonic stem cells (hESCs) have the capability to generate functional hemato-endothelial cells and other mesoderm lineage cells. hESC-derived CD73⁺ cells have been isolated and found to have similar phenotypic and functional characteristics as adult MSCs. Here we demonstrate hESC-derived CD34⁺CD73^? cells can serve as MSC progenitor cells with the ability to differentiate into adipocytes, osteoblasts and chondrocytes. Additionally, gene array analysis of hESC-derived MSCs show substantially different gene expression compared to bone marrow (BM)-derived MSCs, especially with increased expression of pluripotent and multipotent stem cell and endothelial cell-associated genes. The isolation of functional MSCs from hESC-derived CD34⁺CD73^? cells provides improved understanding of MSC development and utilization of pluripotent stem cells to produce MSCs suited for novel regenerative therapies.     

Adipose tissue is an ideal stem cell source for tissue reconstruction of bone，cartilage，and soft tissue defects

目的 验证人脂肪基质细胞是否具有向成骨细胞、软骨细胞、脂肪细胞分化的能力,从而为骨、软骨、软组织再建寻找一种理想的干细胞来源.方法 分别用成骨向分化培养基（DMEM＋10?S＋地塞米松＋维生素C＋β-甘油磷酸）、软骨向分化培养基（DMEM＋1?S＋胰岛素＋维生素C＋转化生长因子β1）及脂肪向分化培养基（DMEM＋10?S＋地塞米松＋胰岛素＋吲哚美辛＋异丁基甲基黄嘌呤）诱导人脂肪基质细胞向成骨细胞、软骨细胞及脂肪细胞分化.用von Kossa和碱性磷酸酶染色鉴定成骨细胞分化,而软骨细胞分化和脂肪细胞分化分别用Alcian blue染色和油红O染色显示.成骨细胞、软骨细胞以及脂肪细胞特异相关或标志基因的表达用RT-PCR检测.结果 体外实验表明,人脂肪基质细胞在定向分化诱导剂的作用下可分别向成骨细胞、软骨细胞及脂肪细胞分化.结论 人脂肪基质细胞中包含有多向分化能力的干细胞,可用于今后骨、软骨、软组织的组织工程再建.     

Muscle derived stem cell therapy for stress urinary incontinence

AIM: The aim of this article is to discuss the potential of muscle-derived stem cells (MDSCs) for rhabdosphincter regeneration and to review the early clinical experiences with its application in patients with stress urinary incontinence. RESULTS: In anatomical and functional studies of the human and animal urethra, the middle urethral contained rhabdosphincter is critical for maintaining continence. Transplanted stem cells have the ability to undergo self-renewal and multipotent differentiation, leading to sphincter regeneration. In addition, such cells may release, or be engineered to release, neurotrophins with subsequent paracrine recruitment of endogenous host cells to concomitantly promote a regenerative response of nerve-integrated muscle. CONCLUSION: Cell-based therapies are most often associated with the use of autologous multipotent stem cells, such as bone marrow stromal cells. However, harvesting bone marrow stromal stem cells requires a general anesthetic, can be painful, and has variable yield of stem cells upon processing. In contrast, with appropriate experience, alternative autologous adult stem cells such as muscle-derived stem cells and adipose-derived stem cells can be obtained in large quantities and with minimal discomfort.     

Osteogenic potential oif rabbit marrow stromal stem cells cultued in vitro：a histochemical and scanning electron microscopic study

Objective:To further investigate the osteogenic potential of rabbit marrow stromalstem cells cultured in vitro.Methods:Rabbit marrow stromal stem cells were isolated by density gradient centrifugation method and amplified in the flasks,using the osteogenic inducing conditions(OGC) as the culture media.The osteogenic potential of marrow stromal stem cells were investigated by means of bone-seeking fluorescenc(tetracycline) labeling, Alizarin red S(ARS) staining,Alcian blue-Sirius red (AS) staining, and scanning electron microscope.Results:After being passaged ,the marrow stromal stem cells increased in number,became confluent and formed multi-layer structure.The stromal stem cells excreted innumerable tiny granules,heaping up on the cell body and merging gradually into foggy substances.These foggy substances kept on enlarging and formed round, oval, or flake-like nodules.These nodules revealed bright golden yellow fluorescence under fluorescence microscope when labeled with tetracycline.Histochemical study with specific new bone staining with ARS revealed positive calcium reaction,both denoting that they were newly formed bone tissues.After they were stained with AS, collagen and acid mucopolysaccharide were shown.Under scanning electron microscope,three types of cells with different configurations were found.They were globular cells,spindle-shaped cells and polygonal or polygonal cells.Granules were excreted from the cells and heaped up on the cell body.Needle-shaped and irregularly rectangular crystals also appeared and agglomerated with the granules to form nodules and trabecula-like or flake-like structures.Conclusions:Sequence of events of bone formation by rabbit marrow stromal stem cells cultured in vitro is fully depicted and confirmed,which provides the foundation for further investigating the mechanisms of osteoblast differentiation from marrow stromal stem cells and the possible application in orthopaedics.