Labeling and tracing of bone marrow mesenchymal stem cells for tendon-to-bone tunnel healing

Purpose  

To investigate the effects of bone marrow mesenchymal stem cells (BMSCs) on tendon-to-bone tunnel healing and provide experimental evidence for labeling and tracing of stem cells.     

Mesenchymal stem cells for the treatment of cartilage lesions: from preclinical findings to clinical application in orthopaedics

Purpose

The aim of this systematic review is to examine the available clinical evidence in the literature to support mesenchymal stem cell (MSC) treatment strategies in orthopaedics for cartilage defect regeneration.

Methods

The research was performed on the PubMed database considering the English literature from 2002 and using the following key words: cartilage, cartilage repair, mesenchymal stem cells, MSCs, bone marrow concentrate (BMC), bone marrow-derived mesenchymal stem cells, bone marrow stromal cells, adipose-derived mesenchymal stem cells, and synovial-derived mesenchymal stem cells.

Results

The systematic research showed an increasing number of published studies on this topic over time and identified 72 preclinical papers and 18 clinical trials. Among the 18 clinical trials identified focusing on cartilage regeneration, none were randomized, five were comparative, six were case series, and seven were case reports; two concerned the use of adipose-derived MSCs, five the use of BMC, and 11 the use of bone marrow-derived MSCs, with preliminary interesting findings ranging from focal chondral defects to articular osteoarthritis degeneration.

Conclusions

Despite the growing interest in this biological approach for cartilage regeneration, knowledge on this topic is still preliminary, as shown by the prevalence of preclinical studies and the presence of low-quality clinical studies. Many aspects have to be optimized, and randomized controlled trials are needed to support the potential of this biological treatment for cartilage repair and to evaluate advantages and disadvantages with respect to the available treatments.

Level of evidence

IV.     

Autologous collagen-induced chondrogenesis technique (ACIC) for the treatment of chondral lesions of the talus

Purpose

Autologous collagen-induced chondrogenesis technique (ACIC) combines microfractures with the use of an injectable atelocollagen matrix that allows performing the whole cartilage repair treatment arthroscopically. The aim of this study was to evaluate the in vitro cytocompatibility of this biomaterial using human bone marrow mesenchymal stem cells and human chondrocytes. Moreover, the preliminary data of five patients affected by chondral lesion of the talus treated with the ACIC technique are shown.

Methods

Human bone marrow mesenchymal stem cells and human chondrocytes were seeded on solid and pre-solid atelocollagen scaffolds. Cell–scaffold constructs were cultured for 7 days and then prepared for histological analyses. Arthroscopic ACIC was performed in five patients affected by chondral lesions of the talus; they were clinically evaluated with AOFAS, VAS and Tegner score before and then after 6 months from surgery.

Results

In vitro results showed that both bone marrow mesenchymal stem cells and chondrocytes were able to efficiently colonize the whole construct, from the surface to the core, only when seeded on the pre-solid atelocollagen scaffold, but not on its solid form. No adverse events were observed in the patients treated with the ACIC technique; a significant improvement in VAS pain scale and in AOFAS score was found at 6 months follow up.

Conclusion

Injectable atelocollagen can be considered a feasible scaffold for cartilage repair treatment, in particular if used in its pre-solid form. ACIC leads to good clinical results in the treatment for chondral lesions of the talus even if longer follow-up and a higher number of patients are necessary to confirm these data.

Level of evidence

IV.     

Effects of bone marrow or mesenchymal stem cell transplantation on oral mucositis (mouse) induced by fractionated irradiation

Background and purpose

Oral mucositis is a severe and dose limiting early side effect of radiotherapy for head-and-neck tumors. This study was initiated to determine the effect of bone marrow- and mesenchymal stem cell transplantation on oral mucositis (mouse tongue model) induced by fractionated irradiation.

Material and methods

Daily fractionated irradiation (5?×?3 Gy/week) was given over 1 (days 0–4) or 3 weeks (days 0–4, 7–11, 14–18). Each protocol was terminated (day 7 or 21) by graded test doses (5 dose groups, 10 animals each) in order to generate complete dose–effect curves. The incidence of mucosal ulceration, corresponding to confluent mucositis grade 3 (RTOG/EORTC), was analyzed as the primary, clinically relevant endpoint. Bone marrow or mesenchymal stem cells were transplanted intravenously at various time points within these fractionation protocols.

Results

Transplantation of 6?×?10⁶, but not of 3?×?10⁶ bone marrow stem cells on day ??1, +?4, +?8, +?11 or +?15 significantly increased the ED₅₀ values (dose, at which an ulcer is expected in 50?% of the mice); transplantation on day +?2, in contrast, was ineffective. Mesenchymal stem cell transplantation on day ??1, 2 or +?8 significantly, and on day +?4 marginally increased the ED₅₀ values.

Conclusion

Transplantation of bone marrow or mesenchymal stem cells has the potential to modulate radiation-induced oral mucositis during fractionated radiotherapy. The effect is dependent on the timing of the transplantation. The mechanisms require further investigation.     

Bone marrow stem cell adherence into old anterior myocardial infarction: a scintigraphic study using Tl-201 and Tc-99m-HMPAO

Aim  

The precise localization of bone marrow stem cells (SCs) into the necrotic tissue after intracoronary infusion (ICI) may be important for the therapeutic outcome. This study aims to examine the correlation between Tl-201 and Tc-99m-hexa-methyl-propylene-amine-oxime (HMPAO) images.     

Is there a role for adult non-cultivated bone marrow stem cells in ACL reconstruction?

Purpose

To assess by magnetic resonance imaging (MRI) if adult non-cultivated bone marrow stem cells accelerate tendon-to-bone healing in the femoral tunnel, after hamstring anterior cruciate ligament (ACL) reconstruction.

Methods

Forty-three patients underwent ACL reconstruction and were prospectively randomized into two groups: 20 patients in the experimental group (group A) with adult non-cultivated bone marrow stem cells and 23 patients in the control group (group B) without adult non-cultivated bone marrow stem cells. All patients underwent MRI of the knee at three months after surgery to evaluate the signal-to-noise ratio of the interzone.

Results

There was no difference in the signal-to-noise ratio of the interzone on MRI between the experimental and the control group.

Conclusions

Adult non-cultivated bone marrow stem cells do not seem to accelerate graft-to-bone healing in ACL reconstruction. The clinical relevance of this finding is that adult non-cultivated bone marrow stem cells apparently have a limited role in ACL reconstruction.

Level of evidence

II.     

Autologous mesenchymal stem cell endografting in experimental cerebrovascular aneurysms

Introduction

Coiling is the gold standard for the treatment of intracranial aneurysms. However, some issues associated with endovascular treatment limit its long-term efficiency. Recanalization with coil compaction is certainly the most important. New approaches may be considered to promote thrombus colonization by mesenchymal cells and aneurysm healing. In the present study, we have percutaneously delivered autologous bone marrow mesenchymal stem cells (BMSCs) to an elastase-induced rabbit carotid aneurysm model in vivo.

Methods

Autologous mesenchymatous stem cells were obtained after femoral puncture and bone marrow aspiration. After 2 weeks of in vitro cell culture, five million BMSCs were grafted in the carotid aneurysm using an endovascular approach.

Results

We demonstrated the feasibility of in vivo percutaneous seeding of autologous BMSCs in the aneurysm by positive Hoechst fluorostaining. Two weeks later, conventional angiography showed an increase in median aneurysmal surface in the sham group, whereas this surface was decreased in the group treated with BMSCs, +28.4 versus ?26.4 %, respectively (p?=?0.01). BMSC seeding resulted in intimal hyperplasia with cell colonization and disappearance of the thrombus.

Conclusion

In conclusion, percutaneous seeding of BMSCs may colonize and heal the arterial wall thus limiting aneurysm expansion.     

MRI of the proximal femur predicts marrow cellularity and the number of mesenchymal stem cells

Purpose:

To determine whether the marrow conversion index (MCI) in MRI is related to the total number of mononuclear and mesenchymal stem cells (MSCs) in proximal femoral metaphysis of patients with hip osteoarthritis.

Materials and Methods:

Thirty‐two hips of 32 consecutive patients who underwent total hip arthroplasty (THA) for hip osteoarthritis were included in this study. MRI of the hip was performed preoperatively and MCI was subsequently calculated. Three‐milliliter bone marrow samples were obtained from the proximal femur during THA and the number of total mononuclear cells was determined using a hemocytometer. Colony forming unit‐fibroblasts (CFU‐Fs) assays of MSCs were performed by transferring a total of 2 × 10⁴ mononuclear cells to each of five 60‐mm plates. One week later, the numbers of colonies were counted.

Results:

The total number of mononuclear cells decreased with increasing MCI. Likewise, the prevalence and total number of CFU‐Fs increased with increasing number of total mononuclear cells, and decreased with increasing MCI.

Conclusion:

Our results suggest that measurement of MCI in MRI can be an objective and noninvasive method to predict marrow cellularity and the number of MSCs in patients with hip osteoarthritis. J. Magn. Reson. Imaging 2012;35:218‐222. © 2011 Wiley Periodicals, Inc.     

Endovascular transplantation of stem cells to the injured rat CNS

Introduction

Transplantation procedures using intraparenchymal injection of stem cells result in tissue injury in addition to associated surgical risks. Intravenous injection of mesenchymal stem cells gives engraftment to lesions, but the method has low efficiency and specificity. In traumatic brain injuries (TBI), there is a transient breakdown of the blood–brain barrier and an inflammatory response, which increase migration of cells from blood to parenchyma. The aim of this investigation was to analyze the effect of intra-arterial administration on cellular engraftment.

Methods

Experimental TBI was produced in a rat model. Endovascular technique was used to administer human mesenchymal stem cells in the ipsilateral internal carotid artery. Evaluation of engraftment and side effects were performed by immunohistochemical analysis of the brain and several other organs. The results were compared to intravenous administration of stem cells.

Results

Intra-arterial transplantion of mesenchymal stem cells resulted in central nervous system (CNS) engraftment without thromboembolic ischemia. We observed a significantly higher number of transplanted cells in the injured hemisphere after intra-arterial compared to intravenous administration both 1 day (p?<?0.01) and 5 days (p?<?0.05) after the transplantation. Some cells were also detected in the spleen but not in the other organs analyzed.

Conclusion

Selective intra-arterial administration of mesenchymal stem cells to the injured CNS is a minimally invasive method for transplantation. The method is significantly more efficient than the intravenous route and causes no side effects in the current model. The technique can potentially be used for repeated transplantation to the CNS after TBI and in other diseases.     

Low-dose and long-term G-CSF treatment can improve severe myocardial ischemia in patients with severe coronary artery disease

Background  

It has been reported that granulocyte colony-stimulating factor (G-CSF) can promote angiogenesis by mobilizing bone marrow stem cells to blood vessels. The purpose of this study is to clarify whether low-dose and long-term G-CSF treatment can improve severe myocardial ischemia.     

Characterization of age-related changes of tendon stem cells from adult human tendons

Purpose

The present study evaluated the presence of stem cells in hamstring tendons from adult subjects of different ages. The aim was to isolate, characterize and expand these cells in vitro, and to evaluate whether cell activities are influenced by age.

Methods

Tendon stem cells (TSCs) were isolated through magnetic sorting from the hamstring tendons of six patients. TSC percentage, morphology and clonogenic potential were evaluated, as well as the expression of specific surface markers. TSC multi-potency was also investigated as a function of age, and quantitative polimerase chain reaction was used to evaluate gene expression of TSCs cultured in suitable differentiating media.

Results

The presence of easily harvestable stem cell population within adult human hamstring tendons was demonstrated. These cells exhibit features such as clonogenicity, multi-potency and mesenchymal stem cells markers expression. The age-related variations in human TSCs affect the number of isolated cells and their self-renewal potential, while multi-potency assays are not influenced by tendon ageing, even though cells from younger individuals expressed higher levels of osteogenic and adipogenic genes, while chondrogenic genes were highly expressed in cells from older individuals.

Conclusions

These results may open new opportunities to study TSCs to better understand tendon physiology, healing and pathological processes such as tendinopathy and degenerative age-related changes opening new frontiers in the management of tendinopathy and tendon ruptures.     

Controllable labelling of stem cells with a novel superparamagnetic iron oxide–loaded cationic nanovesicle for MR imaging

Objective

To investigate the feasibility of highly efficient and controllable stem cell labelling for cellular MRI.

Methods

A new class of cationic, superparamagnetic iron oxide nanoparticle (SPION)-loaded nanovesicles was synthesised to label rat bone marrow mesenchymal stem cells without secondary transfection agents. The optimal labelling conditions and controllability were assessed, and the effect of labelling on cell viability, proliferation activity and multilineage differentiation was determined. In 18 rats, focal ischaemic cerebral injury was induced and the rats randomly injected with 1?×?10⁶ cells labelled with 0-, 8- or 20-mV nanovesicles (n?=?6 each). In vivo MRI was performed to follow grafted cells in contralateral striata, and results were correlated with histology.

Results

Optimal cell labelling conditions involved a concentration of 3.15?μg Fe/mL nanovesicles with 20-mV positive charge and 1-h incubation time. Labelling efficiency showed linear change with an increase in the electric potentials of nanovesicles. Labelling did not affect cell viability, proliferation activity or multilineage differentiation capacity. The distribution and migration of labelled cells could be detected by MRI. Histology confirmed that grafted cells retained the label and remained viable.

Conclusion

Stem cells can be effectively and safely labelled with cationic, SPION-loaded nanovesicles in a controllable way for cellular MRI.

Key Points

? Stem cells can be effectively labelled with cationic, SPION-loaded nanovesicles. ? Labelling did not affect cell viability, proliferation or differentiation. ? Cellular uptake of SPION could be controlled using cationic nanovesicles. ? Labelled cells could migrate along the corpus callosum towards cerebral infarction. ? The grafted, labelled cells retained the label and remained viable.     

成人骨髓间充质干细胞培养上清对人脐血间充质干细胞体外培养及扩增的支持作用

目的：研究人骨髓间充质干细胞培养上清对人脐带血（HUCB）中所含有间充质干细胞（MSCs）的影响。方法：取脐带血，肝素抗凝，Percoll淋巴细胞分离液分离出单个核细胞，低糖DMEM培养获得贴壁细胞层。与人骨髓间充质干细胞培养上清共孵育。流式细胞仪检测表面抗原。结果：脐带血的单个核细胞与人骨髓间充质干细胞培养上清共孵育经体外培养贴壁后出现形似纤维状细胞形态并表达与MSCs相关的抗原（CD13，CD44，CD71，CD166）。但不表达造血细胞抗原（CD34，CD45），这与源于骨髓的MSCs一致。结论：人脐血间充质干细胞与骨髓间充质干细胞培养上清共孵育，对脐血间充质干细胞体外分离培养及扩增有支持作用。     

Conventional rotator cuff repair complemented by the aid of mononuclear autologous stem cells

Purpose  

To investigate the behavior of rotator cuff tears treated with conventional repair technique with the aid of autologous bone marrow mononuclear cells (BMMC).     

In vivo MR imaging tracking of transplanted mesenchymal stem cells in a rabbit model of acute peripheral nerve traction injury

Purpose

To investigate in vivo MRI tracking mesenchymal stem cells (MSCs) in peripheral nerve injures using a clinically available paramagnetic contrast agent (Gd‐DTPA) and commercially available rhodamine‐incorporated transfection reagents (PEI‐FluoR).

Materials and Methods

After bone marrow MSCs were labeled with Gd‐DTPA and PEI‐FluoR complex, the labeling efficacy and longevity of Gd‐DTPA maintenance were measured and cell viability, proliferation, and apoptosis were assessed. Thirty‐six rabbits with acute sciatic nerve traction injury randomly received 1 × 10⁶ labeled (n = 12) or unlabeled MSCs (n = 12) or vehicle alone injection. The distribution and migration of implanted cells was followed by MRI and correlated with histology. The relative signal intensity (RSL) of the grafts was measured.

Results

The labeling efficiency was 76 ± 4.7% and the labeling procedure did not in?uence cell viability, proliferation, and apoptosis. A persistent higher RSL in grafts was found in the labeled group compared with the unlabeled and vehicle groups until 10 days after transplantation (P < 0.05). The distribution and migration of labeled cells could be tracked by MRI until 10 days after transplantation. Transplanted MSCs were not found to transdifferentiate into Schwann‐like cells within 14‐day follow‐up.

Conclusion

Labeling MSCs with the dual agents may enable cellular MRI of the engraftment in the experimental peripheral nerve injury. J. Magn. Reson. Imaging 2010;32:1076–1085. © 2010 Wiley‐Liss, Inc.

     

Human bone marrow mesenchymal stem cells expressing SDF-1 promote hematopoietic stem cell function of human mobilised peripheral blood CD34+ cells in vivo and in vitro

Purpose:?There is mounting evidence demonstrating that stromal cell derived factor-1 (SDF-1) plays an important role in homing of hematopoietic progenitor cells to bone marrow. This study was aimed to assess whether bone marrow mesenchymal stem cells overexpressing exogenous SDF-1 could synergistically promote the homing of CD34⁺ (Cluster of Differentiation [CD]) cells to bone marrow of lethally irradiated severe combined immunodeficiency (SCID) mice.

Methods:?Human SDF-1 complementary Deoxyribonucleic acid (cDNA) was transfected into bone marrow-derived mesenchymal stem cells with recombinant lentiviral vector. The expression of SDF-1 was detected by real-time Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA), and the ex vivo chemotaxis function on CD34⁺ cells was measured by coculture system and Transwell system. SDF-1 gene-modified mesenchymal stem cell (MSC) and CD34⁺ cells were infused into lethally irradiated SCID mice and the hematopoietic reconstitution in the recipient mice was examined.

Results:?Messenger ribonucleic acid (mRNA) and protein of SDF-1 in infected MSC were significantly higher than that of the non-infected control MSC (p?<?0.05). The infected MSC have significant chemotaxis effect on CD34⁺ cells in?vitro and promote hematopoietic reconstitution after CD34⁺ cell transplantation in?vivo.

Conclusion:?MSC with high-level expression of SDF-1 can synergistically promote hematopoietic reconstitution after CD34⁺ cell transplantation in lethally irradiated SCID mice.     

自体骨髓间充质干细胞移植治疗脑瘫2年随访

 目的 观察自体骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)转化神经干细胞治疗脑瘫的临床效果。方法 选择2010-06至2011-06期间, GMFCS级别III～Ⅴ级,自愿接受干细胞移植的脑瘫患儿11例为移植组,同时选择年龄、性别及疾病严重程度相仿的11例脑瘫患儿为对照组。移植组采用hMSCs转化神经干细胞移植治疗和运动功能锻炼为主的康复治疗,对照组仅仅给予运动锻炼为主的康复治疗。采用脑瘫患儿粗大运动功能评定(GMFM)量表评价移植组和对照组在治疗前和治疗后1、3、6、12、18、24个月的GMFM得分。结果 (1)移植组治疗后1个月、3个月、6个月的GMFM较GMFM0上升率均明显高于对照组(P=0.025);移植组治疗后3个月GMFM3较1个月GMFM1的上升率比较有明显差异,而治疗后6个月GMFM6较3个月GMFM3的上升率比较无统计学差异,提示移植组运动能力在移植后2~3个月时快速显示明显的康复效果。对照组GMFM上升率无此特征;(2)随访12个月、18个月及24个月发现移植组和对照组的GMFM12、GMFM18、GMFM24较之前6个月的GMFM的上升率无差异,而较治疗之前的GMFM0的上升率有显著差异(P<0.01)。(3)两组治疗后1个月、3个月的语言商与治疗前语言商比较,上升率差异均无统计学意义,两组治疗后6个月、12个月移植组语言商较治疗前上升率明显高于对照组(P<0.01)而18个月及24个月随访提示语言商上升率较对照组无增快显现,但较治疗前语言商上升率有明显差异(P<0.05)。结论 hMSCs转化神经干细胞移植治疗脑瘫安全,有效,对脑瘫患儿的运动功能改善明显,在移植后3个月明显显效。6个月以后运动康复效果逐渐消失,但患儿运动能力无倒退现象。语言功能的改善显效较晚,在移植治疗后6个月后显效,12个月后语言快速改善效果消失,但没有语言能力的倒退。
     

Non-invasive tracking of human haemopoietic CD34+ stem cells in vivo in immunodeficient mice by using magnetic resonance imaging

Objective

To assess migration of CD34⁺ human stem cells to the bone marrow of athymic mice by using magnetic resonance (MR) imaging and Resovist, a contrast agent containing superparamagnetic iron oxide (SPIO) particles.

Methods

All animal and human procedures were approved by our institution’s ethics committee, and women had given consent to donate umbilical cord blood (UCB). Balb/c-AnN Foxn1^nu/Crl mice received intravenous injection of 1?×?10⁶ (n?=?3), 5?×?10⁶ (n?=?3) or 1?×?10⁷ (n?=?3) human Resovist-labelled CD34⁺ cells; control mice received Resovist (n?=?3). MR imaging was performed before, 2 and 24 h after transplantation. Signal intensities of liver, muscle and bone marrow were measured and analysed by ANOVA and post hoc Student’s t tests. MR imaging data were verified by histological and immunological detection of both human cell surface markers and carboxydextran-coating of the contrast agent.

Results

CD34⁺ cells were efficiently labelled by Resovist without impairment of functionality. Twenty-four hours after administration of labelled cells, MR imaging revealed a significant signal decline in the bone marrow, and histological and immunological analyses confirmed the presence of transplanted human CD34⁺ cells.

Conclusion

Intravenously administered Resovist-labelled CD34⁺ cells home to bone marrow of mice. Homing can be tracked in vivo by using clinical 1.5-T MR imaging technology.     

改良直接贴壁法体外分离培养兔骨髓间充质干细胞及其鉴定

目的 观察并鉴定兔骨髓间充质干细胞生物特性,建立一套简单、高效的培养方法.方法 采用直接贴壁法分离培养兔骨髓间充质干细胞,倒置显微镜下观察其形态.MTT法测定其增殖水平并绘制其生长曲线.流式细胞仪测定第三代骨髓间充质干细胞表型.结果 细胞培养第5d,镜下见细胞呈小集落生长,形态呈多边形;7～8 d小集落融合成大集落,10d左右可传代.第二代细胞镜下呈梭形成纤维样细胞,即间充质干细胞;细胞生长曲线成S形;流式细胞仪检测细胞表达CD 73、CD 90、CD 105、不表达CD 34、CD 45.结论 改良直接贴壁法可获得大量、纯化、稳定的间充质干细胞.