Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells

Supraphysiological concentrations of exogenous growth factors are typically required to obtain bone regeneration, and it is unclear why lower levels are not effective. We hypothesized that delivery of bone progenitor cells along with appropriate combinations of growth factors and scaffold characteristics would allow physiological doses of proteins to be used for therapeutic bone regeneration. We tested this hypothesis by measuring bone formation by rat bone marrow stromal cells (BMSCs) transplanted ectopically in SCID mice using alginate hydrogels. The alginate was gamma-irradiated to vary the degradation rate and then covalently modified with RGD-containing peptides to control cell behavior. In the same delivery vehicle, we incorporated bone morphogenetic protein-2 (BMP2) and transforming growth factor-beta3 (TGF-beta3), either individually or in combination. Individual delivery of BMP2 or TGF-beta3 resulted in negligible bone tissue formation up to 22 weeks, regardless of the implant degradation rate. In contrast, when growth factors were delivered together from readily degradable hydrogels, there was significant bone formation by the transplanted BMSCs as early as 6 weeks after implantation. Furthermore, bone formation, which appeared to occur by endochondral ossification, was achieved with the dual growth factor condition at protein concentrations that were more than an order of magnitude less than those reported previously to be necessary for bone formation. These data demonstrate that appropriate combinations of soluble and biomaterial-mediated regulatory signals in cell-based tissue engineering systems can result in both more efficient and more effective tissue regeneration.     

骨髓基质干细胞在损伤脊髓内向少突胶质细胞定向分化的研究

目的 探讨移植骨髓基质干细胞(BMSCs)在损伤脊髓内向少突胶质细胞分化的可能性.方法 应用低温包埋免疫电镜技术观察迁移在损伤脊髓内1、3、5周的移植BMSCs超微结构,应用免疫荧光标记和激光共聚焦技术观察迁移在损伤脊髓内1、3、5周的移植BMSCs表达髓磷脂碱性蛋白(MBP)、髓鞘蛋白前脂蛋白(PLP)的情况.结果 移植1周,迁移在损伤脊髓白质内的BMSCs体积较小,突起少,细胞核较小,核仁清楚,染色质分布尚均匀,线粒体、粗面内质网和核糖体等细胞器发达,具有少突胶质细胞的超微结构特点;移植3周和5周,迁移在损伤脊髓白质内的BMSCs具有成熟少突胶质细胞的超微结构特点,并形成髓鞘样结构.移植1周后,迁移在损伤脊髓白质内的BMSCs开始表达MBP和PLP;移植3周和5周后,迁移在损伤脊髓白质内的BMSCs继续表达MBP和PLP.结论 移植BMSCs在损伤脊髓内可能会部分分化为功能性少突胶质细胞.     

Arthroplasty of the lunate using bone marrow mesenchymal stromal cells

Mesenchymal stromal cells have the potential to differentiate into a variety of mesenchymal tissues such as bone, cartilage and ligaments. The potential for the regeneration of bone with cartilage coverage has still not been achieved. We evaluated the ability of bone marrow mesenchymal stromal cells to regenerate osteochondral defects in the cavity of the lunate in an animal model. Autologous mesenchymal stromal cells were harvested from the iliac crest of New Zealand white rabbits and expanded in vitro. Total lunate excision was performed in 24 animals and the isolated cells were loaded onto scaffolds. Cell-free scaffolds were implanted in the lunate space of the right wrists of all animals, and the left lunate spaces were filled with predifferentiated, cell-loaded scaffolds. Radiographic and histological analyses were performed after two, six and 12 weeks. In addition, the animals were injected with a fluorescent agent every five days, starting at day 30. After two and six weeks there was no radiographic evidence of ossification, whereas after 12 weeks all animals showed radiographic evidence of ossification. Histological sections showed increasing evidence of cartilage-like cell formation at the edges and new bone tissue in the centre of the newly formed tissue in all groups. The histological examinations showed that bone tissue was located around the newly incorporated vascularisation. This study demonstrated that newly formed vascularisation is necessary for the regeneration of bone tissue with cell-loaded scaffolds.     

Cranial repair using BMP-2 gene engineered bone marrow stromal cells

BACKGROUND: Bone grafts, allografts, and biocompatible artificial bone substitutes all have their shortcomings when used for the repair of cranial bone defects. Tissue engineered bone shows promise as an alternative for the repair of these defects. MATERIALS AND METHODS: Rabbit bone marrow mesenchymal stromal cells (MSCs) were separated from iliac crest aspirates and expanded in a monolayer culture 1 month before implantation. These MSCs were then infected with replication-defective adenovirus-human BMP-2 genes 1 week before implantation. Bilateral critical-size cranial defects were created in the animal with removal of osteoinductive periosteum and dura. MSCs were mixed with alginate UP (ultrapure) to form MSC/polymer construct. MSCs used for the control site were infected with adenovirus beta-galactosidase (beta-gal). After 1 week, 6 weeks, and 3 months, five rabbits from each experimental group were sacrificed and the cranial defect site was examined by histology study. RESULTS: Near-complete repair of the large size cranial defects using the tissue engineered MSC/alginate construct was observed. The H&E stain and von Kossa's staining should better regenerate bone at the experiment site. A statistically significant difference in bone formation was noted by 3D CT imaging at 3 months post-BMP-2 treatment of the cranial defects (0.79 +/- 0.06 versus 0.47 +/- 0.05 cm(2), P < 0.001) but not at 6 weeks (0.36 +/- 0.04 versus 0.33 +/- 0.03 cm(2), P = 0.347). CONCLUSIONS: Near-complete repair of large cranial defects can be achieved using tissue engineered bone. The use of newly developed polymers as well as the integration of the stem cell concept with gene medicine is necessary to attain this goal.     

Repair of large segmental bone defects using bone marrow stromal cells with demineralized bone matrix

Objective: The aim of the present study was to evaluate the effect of tissue‐engineered constructs on repair of large segmental bone defects in goats. Methods: Allogenic demineralized bone matrix (aDBM) was seeded with autologous marrow stromal cells (aMSC) for seven days to construct DBM–MSC grafts prior to implantation. 24 goats were randomly divided into three groups (eight in each). In each group, 3 cm diaphyseal femoral defects were created unilaterally, and subsequently filled with the DBM‐MSC grafts, DBM alone and an untreated control, respectively. Radiological analysis and biomechanical evaluation were performed at 12 and 24 weeks after operation. Results: Obvious increases in radiological scoring and biomechanical strength were found in the DBM‐MSC group when compared to the DBM group. X‐ray examination showed excellent bone healing in the DBM‐MSC group, whereas only partial bone repair was seen in the DBM group, and no healing in untreated controls. Histologically, a tendency to bone regeneration and remodeling was far more obvious for the DBM‐MSC group than the DBM only and untreated controls. Conclusion: Our results strongly suggest that transplantation of bone MSC within a DBM could have advantages for the bone repair of large segmental defects.     

Bone formation following transplantation of genetically modified primary bone marrow stromal cells.

Bone marrow stromal cells contain mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues; in the presence of BMP-2, for example, they differentiate into osteoblasts. We constructed replication-deficient adenoviral vectors encoding human BMP-2 (BMP-2/Ad) or BMP-4 (BMP-4/Ad) and used them to transduce primary bone marrow stromal cells from the femurs of four-week-old female C3H mice, which then expressed and processed functional BMP-2 or BMP-4 protein. Enzyme assays and histochemical staining showed both groups of cells to possess alkaline phosphatase activity, a marker of differentiation into osteoblasts, though the activity was higher in cells transduced with BMP-2/Ad. When BMP-2/Ad-transduced cells were injected into the thigh muscles of immunocompetent C3H mice, ossicle development was detected on radiographs within four weeks after injection. Moreover, histological analysis indicated that newly developed ossicles contain mature osseous components, including cortical bone and bone marrow, within eight weeks. Thus, syngeneic transplantation of genetically modified primary bone marrow stromal cells induced bone formation in immunocompetent mice, perhaps indicating its potential for use in the development of therapeutic protocols aimed at enhancing bone formation.     

In vivo bioluminescence imaging of transplanted bone marrow mesenchymal stromal cells using a magnetic delivery system in a rat fracture model

For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.     

骨髓基质干细胞在体外向软骨细胞分化

目的观察骨髓基质干细胞(B M SC s)在体外能否分化为软骨细胞。方法利用高密度细胞球培养体系在含转化生长因子-β1(TG F-β1)的培养基中培养B M SC s21d,用免疫组化甲苯胺蓝染色方法分析培养的B M SC s球中蛋白多糖(软骨细胞分泌的主要基质成份)的表达、用免疫组化和R T-P C R方法分析Ⅱ型胶原(软骨细胞特异分泌的主要胶原蛋白)的表达来评估B M SC s是否分化为软骨细胞。结果TG F-β1作用的B M SC s表达了Ⅱ型胶原和蛋白多糖。结论B M SC s在体外特定的培养条件下可分化为软骨细胞,从而可能成为临床上治疗创伤或骨关节炎所致的软骨缺损所需的合适的自体来源的种子细胞。     

自体骨髓基质干细胞在齿槽裂骨缺损修复中的应用

目的探讨人自体骨髓基质干细胞（human bone marrow stromal cells，hBMSCs）在治疗齿槽裂骨缺损中的可行性。方法2002至2005年，选择齿槽裂骨缺损患者7例（单侧6例，双侧1例），以患者自体骨髓基质干细胞为种子细胞，部分脱钙骨（partly demineralized bone matrix，pDBM）为支架材料构建组织工程骨，治疗齿槽裂骨缺损。从患者髂前上棘穿刺取骨髓，密度梯度离心法分离hBMSCs，经体外成骨诱导和扩增至第3代。将诱导的hBMSCs，复合部分脱钙骨体外培养1周后，手术回植骨缺损区。分别于术后1、3、6、12、24、36个月进行临床外形和三维CT检查随访。结果6例患者头部三维CT检查，结果示术后3个月能形成组织工程化骨，并修复骨组织缺损。术后1～3年的随访表明组织工程骨稳定存在，无明显骨吸收现象，临床治疗效果稳定。1例患者（双侧齿槽裂）植入物外露感染。结论以自体hBMSCs为种子细胞，部分脱钙骨为支架材料，利用组织工程技术可在人体内形成稳定的组织工程化骨组织，并临床修复齿槽裂骨缺损。     

Effect of serum on human bone marrow stromal cells: ex vivo expansion and in vivo bone formation

BACKGROUND: Bone marrow stromal cell (BMSC) transplantation may offer an efficacious method for the repair of bone defects. This approach has been developed using BMSCs expanded ex vivo in medium with fetal bovine serum (FBS). For clinical applications, however, contact of BMSCs with FBS should be minimized. We studied the effect of FBS substitutes on both human BMSC proliferation in vitro and subsequent bone formation in vivo. METHODS: BMSC proliferation was measured by colony forming efficiency (CFE) and by cell numbers at consecutive passages. Bone formation was studied in 6- to 8-week-old transplants of human BMSCs in immunocompromised mice. RESULTS: Medium with FBS was more effective in stimulating BMSC proliferation than medium with either human serum (HS) or rabbit serum (RS). Compared to bone formed by BMSCs cultured continuously with FBS, bone formed by cells cultured with HS, or with FBS switched to HS, was considerably less extensive, while bone formed by cells cultured with FBS switched to serum-free medium (SFM) was considerably more extensive. The increase in bone formation was due to neither the SFM components nor to the proliferation status of BMSCs prior to transplantation. CONCLUSIONS: Our data demonstrate that for ex vivo expansion of human BMSCs, medium with FBS remains most effective. However, incubation of human BMSCs in SFM prior to in vivo transplantation significantly stimulates subsequent bone formation. This finding increases the practicality of using culture-expanded BMSCs for autologous human transplantation and suggests the presence of osteogenic inhibitors in serum.     

骨髓基质细胞向神经细胞分化研究进展

自2000年美国《科学》杂志首次报道来源于骨髓的细胞进入脑并表达神经表型以来,在动物模型上进行的骨髓基质细胞移植模拟治疗中风、脊髓损伤、肌萎缩性侧索硬化及帕金森病等的研究均显示有一定的疗效.近年来体外诱导骨髓基质细胞向神经系细胞分化的研究颇多,其中涉及到多种生物技术.该文就化合物诱导及潜在问题、蛋白及基因转染问题、骨髓基质细胞与神经细胞共培养等研究作一综述.     

Homocysteine enhances apoptosis in human bone marrow stromal cells

INTRODUCTION: High plasma homocysteine (Hcy) levels have been associated with increased risk of fracture. Since Hcy has been shown to induce apoptosis in many cell types, including vascular endothelial cells, we hypothesized that Hcy would have a similar apoptotic effect on osteoblasts, leading to osteoporosis by reducing bone formation. MATERIALS AND METHODS: Using primary human bone marrow stromal cells (hBMSC) and HS-5 cell line (human bone marrow stromal cell line), we investigated the effects of Hcy on these cells by cell viability assay and analysis of cytoplasmic histone-associated DNA fragments. Caspase activity assay, Western blots, and electrophoresis mobility shift assay (EMSA) were performed to find the mechanism of apoptosis. Intracellular reactive oxygen species (ROS) were measured by spectrometry using dichlorofluorescein diacetate, and cellular total glutathione level was determined by a commercially available kit. N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) were used as tools for investigating the role of ROS and nuclear factor-kappaB (NF-kappaB), respectively. RESULTS: Hcy induced apoptosis in primary human bone marrow stromal cells and the HS-5 cell line, and this apoptotic effect was caspase-dependent. In addition, Hcy increased cytochrome c release into the cytosol, and activated caspase-9 and caspase-3, but not caspase-8, indicating that Hcy induces apoptosis via the mitochondria pathway. Hcy increased ROS, and NAC inhibited the apoptotic effect of Hcy. Western blot and EMSA showed that Hcy activated the NF-kappaB pathway. PDTC blocked Hcy-induced caspase-3 activation and apoptosis. CONCLUSION: These results suggest that Hcy induces apoptosis via the ROS-mediated mitochondrial pathway and NF-kappaB activation in hBMSCs, and that Hcy may contribute to the development of osteoporosis by reducing bone formation. Antioxidants may have a role in preventing bone loss in individuals with hyperhomocysteinemia.     

Restoration of bone defect and enhancement of bone ingrowth using partially demineralized bone matrix and marrow stromal cells

PURPOSE: This study aimed to investigate the capability of combining marrow stromal cells (MSC) and partially demineralized bone matrix (PDBM) to fill bone defect and enhance bone ingrowth using a canine non-weight-bearing gap model. METHODS: Custom-made implants with 3mm gap between the porous surface and the host bone were used. The implants were inserted into the distal femurs of 25 mongrel dogs and the gaps were randomly assigned to be filled with culture-expanded autologous MSC-loaded PDBM, autograft, fresh-frozen allograft, PDBM alone, or nothing as controls. Histomorphometry using backscattered scanning electron microscopic examination, and mechanical push-out test were performed at 6 months after surgery. RESULTS: Histomorphometry showed that amounts of bone regeneration in the gap and bone ingrowth into the porous-coated surface in the MSC-loaded PDBM-treated group were comparable to those of autograft-treated group and were significantly greater than those of allograft-treated, PDBM-treated, or non-grafted groups. Mechanical test showed the same differences. CONCLUSION: The results of this study showed that combining PDBM and autologous culture-expanded MSC restored bone stock and enhanced bone ingrowth into the porous-coated area in a canine non-weight-bearing gap model. This combination may provide an option for reconstructing bone defect when we perform a cementless revision arthroplasty.     

Glucocorticoids impair bone formation of bone marrow stromal stem cells by reciprocally regulating microRNA-34a-5p

Summary

The inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. We found that microRNA-34a-5p was a critical player in dexamethasone (Dex)-inhibited BMSC proliferation and osteogenic differentiation. MicroRNA-34a-5p might be used as a therapeutic target for GC-impaired bone formation.

Introduction

The inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. The mechanisms of this process are still not completely understood. Recent studies implicated an important role of microRNAs in GC-mediated responses in various cellular processes, including cell proliferation and differentiation. Therefore, we hypothesized that these regulatory molecules might be implicated in the process of GC-decreased BMSC proliferation and osteoblastic differentiation.

Methods

Western blot, quantitative real-time PCR, and cell proliferation and osteoblastic differentiation assays were employed to investigate the role of microRNAs in GC-inhibited BMSC proliferation and osteoblastic differentiation.

Results

We found that microRNA-34a-5p was reciprocally regulated by Dex during the process of BMSC proliferation and osteoblastic differentiation. Furthermore, we confirmed that microRNA-34a-5p was a critical player in Dex-inhibited BMSC proliferation and osteogenic differentiation. Mechanistic studies showed that Dex inhibited BMSC proliferation by microRNA-34a-5p targeting cell cycle factors, including CDK4, CDK6, and Cyclin D1. Furthermore, downregulation of microRNA-34a-5p by Dex leads to Notch signaling activation, resulting in inhibition of BMSC osteogenic differentiation.

Conclusions

These results showed that microRNA-34a-5p, a crucial regulator for BMSC proliferation and osteogenic differentiation, might be used as a therapeutic target for GC-impaired bone formation.

     

种植骨髓基质细胞的骨组织工程学研究

目的 观察骨髓基质细胞在多孔状的人工骨块上三维立体培养后的生长情况及其复合植入体内后的成骨能力。方法 利用组织工程学方法,将骨髓基质细胞种植于羟基磷灰石人工骨块上,立体培养２周,用扫描电镜观察细胞在体外的生长情况;将上述细胞人工骨复合体自体异位植入体内,取材观察其植入体内后的成骨情况。结果 细胞在人工骨块上能立体培养成活,细胞多生长于周边的孔隙表面,尤其以贴近培养瓶底的那一边较多,骨块的中心部位未     

In vivo fluorescence tracking of bone marrow stromal cells transplanted into a pneumatic injury model of rat spinal cord

Recent experimental studies have shown that bone marrow stromal cells (BMSC) differentiate into neural cells and reduce neurological deficits when transplanted into traumatized spinal cord. These findings have been derived primarily from histological analyses. We conducted a study directed chiefly at developing a non-invasive system for tracking BMSC transplanted into the spinal cord of living animals. In this study, we induced spinal cord injury (SCI) in rats with a pneumatic device. BMSC were harvested from transgenic mice expressing green fluorescence protein (BMSC-GFP), and were transplanted stereotactically into a control group of rats without SCI (n = 6) and a group with SCI (n = 3). At 2 and 4 weeks after transplantation, the dura mater was exposed and green fluorescence derived from the transplanted BMSC-GFP was observed. The distribution and differentiation of the transplanted cells were subsequently evaluated with immunohistochemistry. Green fluorescence could be detected around the transplantation site in three of six of the control rats. In all three rats subjected to SCI, green fluorescence was shown to spread from the site of BMSC-GFP injection toward the injury site, suggesting that the transplanted cells had migrated toward the lesion within the 4-week post-transplantation period. Histological evaluation suggested that the detected green fluorescence was emitted by cells that had distributed in the dorsal white matter, and demonstrated that some of the transplanted cells expressed neuronal or astrocytic markers. These results suggest the possibility of tracking BMSC transplanted into the spinal cord in living animals. Such noninvasive bioimaging techniques would be valuable for monitoring the fate of these transplanted cells and assessing the safety and efficacy of their transplantation.