Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells

To explore the feasibility of repairing clinical cutaneous deficiency, autogenic bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into epidermal cells and fibroblasts in vitro supplemented with different inducing factors and biomaterials to construct functional tissueengineered skin. The results showed that after 72 h induction, BMSCs displayed morphologic changes such as typical epidermal cell arrangement, from spindle shape to round or oval; tonofibrils, melanosomes and keratohyaline granules were observed under a transmission electronic microscope. The differentiated cells expressed epidermal stem cell surface marker CK19 (59.66% ± 4.2%) and epidermal cells differentiation marker CK10. In addition, the induced epidermal cells acquired the anti-radiation capacity featured by lowered apoptosis following exposure to UVB. On the other hand, the collagen microfibrils deposition was noticed under a transmission electronic microscope after differentiating into dermis fibroblasts; RT-PCR identified collagen type I mRNA expression in differentiated cells; radioimmunoassay detected the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (up to 115.06 pg/mL and 0.84 ng/mL, respectively). Further in vivo implanting BMSCs with scaffold material shortened skin wound repair significantly. In one word, autogenic BMSCs have the potential to differentiate into epidermal cells and fibroblasts in vitro, and show clinical feasibility acting as epidermis-like and dermis-like seed cells in skin engineering.     

Local signals in stem cell-based bone marrow regeneration

The cellular basis of bone marrow （BM） tissue development and regeneration is mediated through hematopoietic stem cells （HSCs） and mesenchymal stem cells （MSCs）. Local interplays between hematopoietic cells and BM stromal cells （BMSCs） determine the reconstitution of hematopoiesis after myelosuppression. Here we review the BM local signals in control of BM regeneration after insults. Hematopoietic growth factors （HGFs） and cytokines produced by BMSCs are primary factors in regulation ofBM hematopoiesis. Morphogens which are critical to early embryo development in multiple species have been added to the family of HSCs regulators, including families of Wnt proteins, Notch ligands, BMPs, and Hedgehogs. Global gene expression analysis of HSCs and BMSCs has begun to reveal signature groups of genes for both cell types. More importantly, analysis of global gene expression coupled with biochemical and biological studies of local signals during BM regeneration have strongly suggested that HGFs and cytokines may not be the primary local regulators for BM recovery, rather chemokines （SDF- 1, FGF-4） and angiogenic growth factors （VEGF-A, Ang- 1） play instructive roles in BM reconstitution after myelosuppression. A new direction of management of BM toxicity is emerging from the identification of BM regenerative regulators.     

Cell-based approaches to the engineering of vascularized bone tissue

This review summarizes recent efforts to create vascularized bone tissue in vitro and in vivo through the use of cell-based therapy approaches. The treatment of large and recalcitrant bone wounds is a serious clinical problem, and in the United States approximately 10% of all fractures are complicated by delayed union or non-union. Treatment approaches with the use of growth factor and gene delivery have shown some promise, but results are variable and clinical complications have arisen. Cell-based therapies offer the potential to recapitulate key components of the bone-healing cascade, which involves concomitant regeneration of vasculature and new bone tissue. For this reason, osteogenic and vasculogenic cell types have been combined in co-cultures to capitalize on the function of each cell type and to promote heterotypic interactions. Experiments in both two-dimensional and three-dimensional systems have provided insight into the mechanisms by which osteogenic and vasculogenic cells interact to form vascularized bone, and these approaches have been translated to ectopic and orthotopic models in small-animal studies. The knowledge generated by these studies will inform and facilitate the next generation of pre-clinical studies, which are needed to move cell-based orthopaedic repair strategies into the clinic. The science and application of cytotherapy for repair of large and ischemic bone defects is developing rapidly and promises to provide new treatment methods for these challenging clinical problems.     

Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.     

Directly auto‐transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto‐transplanted versus in vitro expanded MSC with or without bone morphogenetic protein‐2 (BMP‐2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto‐transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT‐PCR analysis before subcutaneous implantation in combination with BMP‐2 and β‐tricalcium phosphate/hydroxyapatite (β‐TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT‐PCR evaluation. Sheep MSC were CD29⁺, CD44⁺ and CD166⁺ after selection by Ficoll gradient centrifugation, while directly auto‐transplanted MSC‐populations expressed CD29 and CD166 at lower levels. Both, directly auto‐transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto‐transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β‐TCP/HA matrix comparable to the application of 60 μg/ml BMP‐2 only or implantation of expanded MSC. Bone matrix proteins were up‐regulated in constructs following direct auto‐transplantation and in expanded MSC as well as in BMP‐2 constructs. Up‐regulation was detected using immunohistology methods and RT‐PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto‐transplanted or expanded MSC with β‐TCP/HA granules alone. Hence BMP‐2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.     

Engineering bone: challenges and obstacles

Repair of large bone defects is still a challenge for the orthopaedic, reconstructive and maxillo-facial surgeon. Availability of pluripotent stem cells from either autologous or allogenic sources and the potential of inducing the osteogenic phenotype is motivating exploration and development of custom-tailored materials known as "bioengineered bone constructs". In such cases, the clinical scenario involves either expansion of stem cells in monolayer and loading them into a porous scaffold prior to surgery or direct cell expansion within the scaffold, and implanting this novel construct back into the donor patient. In this review, we delineate, from an engineering perspective, the progress that has been made to date and the challenges remaining in successfully translating this promising (but not yet definitively established) approach from bench to the bed site.     

骨髓间充质干细胞生物学特性及其在牙周组织工程的应用

牙周病是一种慢性进行性疾病,可导致牙周支持组织的破坏,最终导致牙丧失。牙周病治疗的目的不仅在于控制炎症,更在于使已经破坏的牙周组织再生以形成新附着。牙周的重建包括因炎症破坏吸收的硬组织(牙槽骨与牙骨质)和软组织(牙周膜与牙龈)。骨髓间充质干细胞有其独特的生物学特性从而作为种子细胞有巨大潜力。以骨髓间充质干细胞作为种子细胞联合组织工程技术应用于牙周组织再生为根治牙周病提供了新思路。     

Timing of transplantation of autologous bone marrow derived mesenchymal stem cells for treating myocardial infarction

It is still unclear whether the timing of intracoronary stem cell therapy affects the therapeutic response in patients with myocardial infarction.The natural course of healing the infarction and the presence of putative homing signals within the damaged myocardium appear to favor cell engraftment during the transendothelial passage in the early days after reperfusion.However,the adverse inflammatory environment,with its high oxidative stress,might be deleterious if cells are administered too early after reperfusion.Here we highlight several aspects of the timing of intracoronary stem cell therapy.Our results showed that transplantation of bone marrow mesenchymal stem cells at 2 4 weeks after myocardial infarction is more favorable for reduction of the scar area,inhibition of left ventricular remodeling,and recovery of heart function.Coronary injection of autologous bone marrow mesenchymal stem cells at 2 4 weeks after acute myocardial infarction is safe and does not increase the incidence of complications.     

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

组织工程中应力对骨髓间充质干细胞分化为成骨细胞的影响

骨髓间充质干细胞具有自我复制、未分化的特点,并可在不同条件下分化为中胚层起源的多种细胞,是一种成体多能干细胞。就组织工程而言,良好的种子细胞是组织工程技术的关键,骨髓间充质干细胞的性质决定了其在骨组织工程领域中的重要地位。此外,骨骼系统属于机体的运动系统,承担体重是骨骼的重要功能之一;而且,人体内几乎所有的细胞都会受到力学因素的影响,故有必要研究力学因素对骨髓间充质干细胞诱导分化为成骨细胞的作用,为骨髓间充质干细胞的体外扩增、诱导分化及培养提供一种新途径。     

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.     

Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z®RP platform

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three‐dimensional functional tissue‐like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three‐dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram®. Furthermore, hydroxyapatite coated Sponceram® was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP‐expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram® compared to hydroxyapatite coated Sponceram®. With this study, we present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

HSV-1感染大鼠骨髓间充质干细胞及形成潜伏感染的初步研究

在体外培养大鼠骨髓间充质干细胞(BMSCs),观察单纯疱疹病毒1型感染骨髓间充质干细胞情况.分离并鉴定BMSCs;HSV-1感染BMSCs,观察细胞病变(CPE);建立BMSCs的HSV-1潜伏感染模型.提取总DNA,PCR法扩增BMSCs内的HSV-1特异性片段,检测HSV-1感染BMSCs及潜伏感染.结果显示骨髓间充质干细胞经14d诱导后,碱性磷酸酶含量增高、形成钙结节,表现出成骨细胞特性.HSV-1感染BMSCs,出现典型的CPE,PCR法证实BMSCs内存在HSV-1的特异性片段.HSV-1潜伏感染的BMSCs,未出现明显的CPE,细胞传至7代,仍可测到HSV-1的基因片段,表明BMSCs有可能形成HSV-1的潜伏感染.大鼠骨髓间充质干细胞在体外可以向成骨细胞方向分化,可作为组织工程学的种子细胞.HSV-1可以在体外感染骨髓间充质干细胞并有形成潜伏感染的趋势.     

Effect of bone sialoprotein on proliferation and osteodifferentiation of human bone marrow-derived mesenchymal stem cells in vitro

We performed this study to investigate the effects of recombinant human bone sialoprotein (BSP) on the proliferation and osteodifferentiation of human BMSCs(hBMSCs). The hBMSC cultures were divided into 4 groups: control group, 10⁻¹⁰ M BSP group (BSP group), osteogenic medium group (10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 mg/L ascorbic acid, OM group) and BSP + OM group (OM plus10⁻¹⁰ M BSP). Compared with the control group, cell growth of the other three groups slowed down, while fluorescence at the G₀/G₁ phase increased. After 28 days, in the OM group and the BSP + OM group, the proportion of STRO-1-positive cells decreased by 22.7% and 38.4% and ALP activity increased by 50% and 71.43%, respectively. CD271 mRNA expression decreased while Cbfa1, osteocalcin and osterix mRNA levels increased in the OM and BSP + OM groups, and the mRNA level change was greater in the BSP + OM group. After 28 days, the number of nodules in the BSP + OM group was 112.5% more than that in the OM group, but nodules did not formed in the control or BSP group. We conclude that BSP is capable of inhibiting hBMSCs proliferation and enhancing their osteogenic differentiation and mineralization in the presence of OM.     

Three-dimensional epidermis-like growth of human mesenchymal stem cells on dermal equivalents: contribution to tissue organization by adaptation of myofibroblastic phenotype and function

Abstract Human mesenchymal stem cells (hMSC) are able to differentiate into mature cells of various mesenchymal tissues. Recent studies have reported that hMSC may even give rise to cells of ectodermal origin. This indication of plasticity makes hMSC a promising donor source for cell-based therapies. This study explores the differentiation potential of hMSC in a tissue-specific microenvironment simulated in vitro . HMSC were cultured air-exposed on dermal equivalents (DEs) consisting of collagen types I and III with dermal fibroblasts and subjected to conditions similar to those used for tissue engineering of skin with keratinocytes. Culture conditions were additionally modified by pre-treating the cells with 5-azacytidine or supplementing the medium with all trans retinoic acid (RA). HMSC were capable of adaptation to epidermis-specific conditions without losing their mesenchymal multipotency. However, despite the viability and evident three-dimensional epidermis-like growth pattern, hMSC showed a persistent expression of mesenchymal but not of epithelial markers, thus indicating a lack of epidermal (trans) differentiation. Further, electron microscopy and immunohistochemical analyses demonstrated that hMSC cultured under epidermis-specific conditions adopted a myofibroblastic phenotype and function, promoted in particular by air exposure. In conclusion, multipotent hMSC failed to differentiate into E-cadherin- or cytokeratin-expressing cells under optimized organotypic culture conditions for keratinocytes but differentiated into myofibroblast-like cells contracting the extracellular matrix, a phenomenon that was enhanced by RA and 5-azacytidine. These results indicate that hMSC might contribute to wound-healing processes by extracellular matrix reorganization and wound contraction but not by differentiation into keratinocytes.     

The pro-metastatic role of bone marrow-derived cells: a focus on MSCs and regulatory T cells

Several bone marrow-derived cells have been shown to promote tumour growth and progression. These cells can home to the primary tumour and become active components of the tumour microenvironment. Recent studies have also identified bone marrow-derived cells—such as mesenchymal stem cells and regulatory T cells—as contributors to cancer metastasis. The innate versatility of these cells provides diverse functional aid to promote malignancy, ranging from structural support to signal-mediated suppression of the host immune response. Here, we review the role of mesenchymal stem cells and regulatory T cells in cancer metastasis. A better understanding of the bipolar nature of these bone marrow-derived cells in physiological and malignant contexts could pave the way for new therapeutics against metastatic disease.     

川芎嗪体外诱导小鼠骨髓间充质干细胞分化为神经元样细胞的研究

为了探讨川芎嗪体外诱导小鼠骨髓间质干细胞（BMSCs）分化为神经元样细胞的作用,以小鼠骨髓间充质干细胞为研究对象,实验分为空白对照组、β-巯基乙醇（BME）阳性对照组和川芎嗪诱导组。采用荧光免疫化学和Western blot方法,分别检测神经干细胞巢蛋白（nestin）和经元特异性烯醇化酶（NSE）的表达;RT-PCR检测诱导不同时间对神经细胞相关基因Nestin、NSE、β-微管蛋白III（β-Tubulin III）和核受体相关因子-1（Nurr1）mRNA表达的影响。结果显示川芎嗪诱导间充质干细胞24 h后,细胞形态发生显著改变,细胞突起形成且数目不等,形成神经元样细胞。细胞死亡率低于β-巯基乙醇诱导组。免疫荧光化学法和western blot结果显示：川芎嗪诱导后的细胞nes-tin和NSE蛋白表达呈阳性,且表达丰度显著高于β-巯基乙醇诱导组。川芎嗪作用不同时间的BMSCs表达神经细胞相关基因Nestin、β-Tubulin III、NSE和Nurrl。结果表明川芎嗪能定向诱导小鼠骨髓间充质干细胞分化为神经元样细胞,是较理想的诱导剂。     

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.