Repair of diaphyseal bone defects with calcitriol-loaded PLGA scaffolds and marrow stromal cells

Calcitriol (1,25(OH)2D3)-loaded porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds prepared by solvent casting/salt leaching method were used to repair a 1.5 cm diaphyseal segmental bone defect as a fully absorbable osteogenic biomaterial. The in vitro release of sulforhodamine B (SRB) from PLGA scaffold was measured using spectrophotometer, considering SRB as a model drug. The SRB released from SRB-incorporated PLGA scaffold during 3 months was with relatively low initial burst. The calcitriol-loaded PLGA scaffolds with or without marrow stromal cells (MSCs) were implanted in a critical-sized intercalated bone defect in rabbit femur. Defects were assessed by radiographs until 9 weeks. The bony union of the defect was observed only in the calcitriol-loaded groups. RT-PCR results indicated that MSCs, which were seeded into calcitriol-loaded scaffold, expressed an increased level of alkaline phosphatase, osteonectin, and type I collagen mRNA at day 10. After 2 and 4 weeks, the implanted scaffolds were evaluated by histology. New osteoid matrix and direct calcium deposits were more evident in calcitriol/PLGA/MSC group. Three-dimensional computed tomography and frontal tomographic images of repaired femur showed that normal femur anatomy had been restored with cortical bone with no implanted PLGA remnants at 20 weeks. It can be concluded that the porous calcitriol-loaded PLGA scaffold combined with MSCs may be a novel method for repairing the large loaded bone defect.     

Tissue engineering of articular cartilage with autologous cultured adipose tissue-derived stromal cells using atelocollagen honeycomb-shaped scaffold with a membrane sealing in rabbits

Adipose tissue derived stromal cells (ATSCs), which were isolated from adipose tissue of rabbit, have shown to possess multipotential, that is, they differentiate into osteoblasts and adipocytes in plate-culturing and into chondrocytes in an established aggregate culture using defined differentiation-inductive medium. The aim of this study was to evaluate the utility of ATSCs in tissue engineering procedures for repair of articular cartilage-defects using the atelocollagen honeycomb-shaped scaffold with a membrane sealing (ACHMS-scaffold). We intended to repair full-thickness articular cartilage defects in rabbit knees using autologously cultured ATSCs embedded in the ACHMS-scaffold. ATSCs were incubated within the ACHMS-scaffold to allow a high density and three-dimensional culture with control medium. An articular cartilage defect was created on the patellar groove of the femur, and the defect was filled with the ATSCs-containing ACHMS-scaffold, ACHMS-scaffold alone, or empty (control). Twelve weeks after the operation, the histological analyses showed that only the defects treated with the ATSCs-containing ACHMS-scaffold were filled with reparative hyaline cartilage, highly expressed Type II collagen. These results indicate that transplantation of autologous ATSCs-containing ACHMS-scaffold is effective in repairing articular cartilage defects.     

Simultaneous injection of bone marrow cells and stromal cells into bone marrow accelerates hematopoiesis in vivo

We have previously demonstrated that stromal cells can support the proliferation and differentiation of hematopoietic cells in vitro and in vivo and that a major histocompatibility complex restriction exists between hematopoietic stem cells and stromal cells. We have also found that intra-bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) leads to more rapid reconstitution of hematopoietic cells than intravenous injection. In the present study, we examine the effect of simultaneous injection of stromal cells and BMCs into the same bone marrow on the recovery of donor hematopoietic cells and demonstrate that simultaneous IBM injection of BMCs plus stromal cells is more effective in reconstituting recipients with donor hematopoietic cells than intravenous injection of BMCs plus stromal cells or IBM injection of BMCs alone.     

A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells

We developed a natural, acellular, 3-D interconnected porous scaffold derived from cartilage extracellular matrix (ECM). Human cartilage was physically shattered, then decellularized sequentially with use of hypotonic buffer, TritonX-100, and a nuclease solution and made into a suspension. The scaffold was fabricated by simple freeze-drying and cross-linking techniques. On histology, scaffolds showed most of the ECM components after removal of the cell fragments, and scanning electron microscopy revealed a 3-D interconnected porous structure. Cellular viability assay revealed no cytotoxic effects. In vitro study showed that the novel scaffold could provide a suitable 3-D environment to support the adheration, proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to chondrocytes in culture with chondrogenic medium after 21 days. Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds and implanted subcutaneously into nude mice. Four weeks later, cartilage-like tissue formed, with positive staining for Safranin O, tuoluidine blue and collagen II. Cells in the samples seemed to confirm that they originated from the labeled BMSCs, as confirmed by in vivo fluorescent imaging and immunofluorescence examination. In conclusion, the cartilage ECM-derived porous scaffold shows potential as biomaterial for cartilage tissue engineering, and PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo.     

Adenovirus-mediated transfer of VEGF into marrow stromal cells combined with PLGA/TCP scaffold increases vascularization and promotes bone repair in vivo

Introduction

Large osseous defect remains a serious clinical problem due to the lack of sufficient blood supply and it has been proposed that this situation can be relieved by accelerating the formation of new vessels in the process of bone defect repair. The aim of this study was to develop a new type of artificial bone by transferring the VEGF gene into marrow stromal cells (MSCs) and seeding them into a porous scaffold.

Material and methods

An adenovirus vector was employed to transfer the VEGF gene into MSCs and expression of the exogenous gene was confirmed by ELISA. Next the transduced cells were seeded into a collagen I modified PLGA/TCP scaffold. The constructed new complex artificial bone was then assessed for biocompatibility in vitro and blood vessel formation and bone formation in vivo.

Results

We found that adenovirus mediated VEGF gene transfer into MSCs sustained VEGF expression in MSCs for 3 weeks. Porous scaffold PLGA/TCP made by rapid prototyping technology exhibited improved biocompatibility resulting from crosslinking with collagen I. Furthermore, the in vivo study showed that large amounts of blood vessels were detected histologically 1 week after artificial bone implantation, and significant bone formation was detected 8 weeks after implantation.

Conclusions

Our findings suggest that gene transfer of VEGF into MSCs combined with PLGA/TCP scaffold enhances bone repair in vivo by promoting vascularization.     

Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl(2). After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process.     

骨髓间充质干细胞复合多肽凝胶及成软骨生成因子修复兔关节软骨缺损

背景：多肽水凝胶因为其具有良好的可塑型性,能够与损伤部位很好的无缝隙结合,所以采用该材料作为支架是骨、软骨组织工程中一种可行的探索。 目的：骨髓间充质干细胞联合新型可注射多肽凝胶及成软骨生成因子修复兔关节软骨缺损,观察其修复效果。 方法：首先分离培养兔骨髓间充质干细胞,兔左侧膝关节处制备直径5 mm,深3 mm的全层骨-软骨缺损模型;右侧造模后空置作为对照。实验分为3组,单纯自组装多肽凝胶移植组,自组装多肽凝胶+成软骨因子组和自组装多肽凝胶+成软骨因子+骨髓间充质干细胞组。采用的成软骨因子包括转化生长因子β1,地塞米松和胰岛素样生长因子1,三者混合后加入到自组装多肽凝胶或骨髓间充质干细胞中。于处理后12周时处死动物行大体及组织学观察、X射线摄片、免疫组织化学法进行组织学评分评估修复情况。 结果与结论：单纯自组装多肽凝胶移植在12周后显示出非常好的修复效果,可见番红O染色,Ⅱ型胶原蛋白免疫组织化学染色强度以及组织学评分明显高于其他组(P < 0.05)。自组装多肽凝胶+成软骨因子组修复效果较好,与自组装多肽凝胶组相似,但其修复区域蛋白聚糖表达比对照组明显升高(P < 0.01)。自组装多肽凝胶+成软骨因子+骨髓间充质干细胞组修复效果不佳,12周未能完全修复缺损区域,与单纯自组装多肽凝胶组比较骨赘的形成有所增加。结果表明,单纯自组装多肽凝胶能够在原位修复骨软骨缺损并促进软骨修复,提示以自组装多肽凝胶支架移植有望提高目前修复软骨缺损的效果。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

丝素蛋白-壳聚糖支架复合骨髓间充质干细胞体内构建组织工程化软骨的生物相容性

文题释义： 生物相容性：是指生命体组织对非活性材料产生的一种性能,一般是指材料与宿主之间的相容性,包括组织相容性和血液相容性。 检测相容性的方法：是将支架材料与种子细胞在体外共培养,检测支架毒性、细胞活性、细胞增殖及细胞与支架的黏附情况等指标,该方法具有客观性强、可重复性强、影响因素相对简单及敏感性高等特点。 背景：课题组前期的研究中发现,丝素蛋白-壳聚糖支架材料复合诱导后骨髓间充质干细胞在兔体内能修复缺损的软骨组织,但对于该组织工程化软骨组织的生物相容性还未进一步研究。 目的：研究丝素蛋白-壳聚糖支架材料复合骨髓间充质干细胞在体内构建组织工程化软骨的生物相容性。 方法：使用丝素蛋白-壳聚糖按1∶1比例混合制备三维支架材料,提取兔骨髓间充质干细胞,将诱导后的骨髓间充质干细胞与丝素蛋白-壳聚糖支架构建修复体,再将修复体移植到兔关节软骨缺损模型中修复软骨组织。实验分为3组,实验组植入诱导后骨髓间充质干细胞+丝素蛋白-壳聚糖支架,对照组植入丝素蛋白-壳聚糖支架干预,空白组未植入修复体。 结果与结论：①实验成功制备丝素蛋白-壳聚糖三维支架材料及提取骨髓间充质干细胞,并构建软骨缺损的修复体,将修复体植入兔体内能成功修复缺损的软骨组织;②建模后2,4,8,12周,3组血常规、降钙素原、血沉、C-反应蛋白结果提示无明显的全身感染征象,3组血常规及肝肾功能各时间段比较差异无显著性意义(P > 0.05);③一般观察、苏木精-伊红染色及扫描电镜观察：建模后12周,相比其他两组,实验组软骨缺损已修复,支架材料已吸收,修复组织周围未见炎性细胞,修复组织已正常组织整合良好;④结果证实,丝素蛋白-壳聚糖支架复合骨髓间充质干细胞在体内构建的组织工程化软骨具有良好的生物相容性。 ORCID: 0000-0002-8139-1175(佘荣峰) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

The measurement of biomechanical properties of porcine articular cartilage using atomic force microscopy

We have recently demonstrated that indentation-type atomic force microscopy (IT-AFM) is capable of detecting early onset osteoarthritis (OA) (Stolz, 2009). This study was based on biopsies, using a desk-top commercial atomic force microscope (AFM). However, cartilage analysis in the knee joints needs to be non-destructive to avoid new seeding points for OA by the taking of biopsies. This requires bringing the probe tip in contact with the articular cartilage (AC) surface inside the joint. Here we present our recent progress towards a medical instrument for performing such IT-AFM measurements for in-vivo knee diagnostics. The scanning force arthroscope (SFA) integrates a miniaturized AFM into a standard arthroscopic sleeve, and is used for direct, quantitative, in situ inspection of AC (Imer et al., 2006). The stabilization and the positioning of the instrument relative to the surface under investigation were performed by means of eight inflatable balloons. An integrated three-dimensional, piezoelectric scanner allowed raster scanning and probing of a small area of cartilage around the point of insertion. An AFM probe with an integrated deflection sensor was mounted at the distal end of the instrument. Using this instrument, several measurements were performed on agarose gel and on porcine cartilage samples. The load-displacement curves obtained were analyzed and the dynamic elastic moduli | E(*) | were calculated. A good correlation between these values and those published in the scientific literature was found. Therefore, we concluded that the SFA can provide quantitative measurements to detect early pathological changes in OA.     

Maxillary sinus floor elevation using a tissue-engineered bone with rhBMP-2-loaded porous calcium phosphate cement scaffold and bone marrow stromal cells in rabbits

The aim of this study was to evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex with recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded porous calcium phosphate cement (CPC) scaffold and bone marrow stromal cells (bMSCs) in rabbits. bMSCs were cultured and osteogenically induced. The osteoblastic differentiation of expanded bMSCs was detected by alkaline phosphatase activity, and calcium deposits in vitro. Thirty-six rabbits were randomly allocated into week 2, 4 and 8 observation groups. At each time point, 24 maxillary sinus floor elevation surgeries in 12 rabbits were performed bilaterally and randomly implanted by (1) CPC materials alone (group A, n = 6), (2) rhBMP-2/CPC composite materials alone (group B, n = 6), (3) CPC/bMSCs complex (group C, n = 6) and (4) rhBMP-2/CPC/bMSCs complex (group D, n = 6). As for maxillary sinus floor elevation, rhBMP-2-loaded CPC could promote new bone formation as compared to CPC, while addition of bMSCs could further enhance its new bone formation and maturity significantly, as detected by histological findings, and fluorochrome labeling. Our data suggested that rhBMP-2/CPC possessed excellent osteoinductive ability, while combining with bMSCs could further promote new bone formation and maturation in maxillary sinus elevation.     

Implant-assisted meniscal repair in vivo using a chondrocyte-seeded flexible PLGA scaffold

A cell-based engineered construct can be used for healing of intractable meniscal lesions. Our aims were to assess the culture conditions (static versus dynamic oscillation) and the healing capacity of the chondrocyte-seeded flexible implants in a heterotopic mouse model. Swine articular chondrocytes were labeled with PKH 26 or DiI dye and seeded onto a flexible PLGA scaffold using dynamic oscillating conditions for 24 h. Half of cell-seeded scaffolds were cultured in the same dynamic conditions, while the remaining scaffolds were cultured statically. After 7 days, scaffolds were placed between swine meniscal discs and were implanted subcutaneously in nude mice for 6 weeks. Additional constructs for assessing in vivo cell tracking were implanted for 12 weeks. Live/dead assays demonstrated labeled chondrocytes attached throughout the scaffold in both culture conditions. DNA measurements showed no significant difference between the culture conditions. A continuous fibro-cartilaginous healing tissue was observed between meniscal discs in all 12 dynamically cultured constructs and 9 of 11 statically cultured ones. There was no evidence of meniscal healing using acellular scaffold as well as in meniscal constructs lacking an implant. Both PKH 26- and DiI-labeled cells were identified along the healing interface. We conclude the chondrocyte-seeded flexible PLGA implants induce healing of meniscal discs in nude mice. Culture conditions after seeding have no apparent effects on healing.     

基因修饰骨髓间充质干细胞修复关节软骨损伤

背景：随着生物技术的发展,通过转基因技术修饰细胞,从而获得长期稳定表达的生物活性因子以治疗关节软骨损伤逐渐引起重视。 目的：就基因修饰的骨髓间充质干细胞在修复关节软骨损伤中的应用作一综述。 方法：由第一作者检索1990至2011年PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed)有关基因修饰骨髓间充质干细胞修复关节软骨损伤的文献,英文检索词为“cartilage,gene therapy,mesenchymal stem cells,tissue engineering,bioactive factor,vector”。共纳入15篇文献归纳总结。 结果与结论：骨髓间充质干细胞已被广泛应用于修复关节软骨损伤。通过转基因技术将特定外源基因导入骨髓间充质干细胞,联合细胞治疗和基因治疗可达到更好的治疗效果,在关节软骨损伤的治疗中有广阔的应用前景。     

Repair of porcine articular osteochondral defects in non-weightbearing areas with autologous bone marrow stromal cells

In vivo niche is known to play important roles in terminal differentiation of implanted bone marrow stromal cells (BMSCs). This study explored the feasibility of repairing articular osteochondral defects using autologous BMSCs and biodegradable polymers. BMSCs from 18 hybrid pigs' marrows were either treated with dexamethasone (40 ng/mL) alone or chondrogenically induced with dexamethasone and transforming growth factor-beta1 (10 ng/mL). The cells were seeded respectively onto polylactic acid (PLA)- coated polyglycolic acid (PGA) scaffolds. Four osteochondral defects in each animal were created at non-weightbearing areas of knee joints (2/each side) and were respectively repaired by a chondrogenically induced BMSC-PGA/PLA construct in experimental group (Exp), by a dexamethasone-treated BMSC-PGA/PLA construct in control 1 group (Ctrl 1), by a PGA/PLA construct alone in control 2 group (Ctrl 2), or left unrepaired in control 3 group (Ctrl 3). To trace the implanted cells, green fluorescent protein (GFP)- labeled BMSCs were implanted in 2 animals. Gross view and histology showed that Exp and Ctrl 1 (with cell implantation) achieved better reparative results than Ctrl 2 and Ctrl 3 (without cell implantation) in terms of the reparative level and the restoration of the histological structure. In addition, 6-month results were better than 3-month results in all 4 groups. In Exp, 11 of 16 defects were completely repaired by hyaline cartilage and cancellous bone. In Ctrl 1, 11 of 16 defects were repaired by fibrocartilage and cancellous bone, although the repair with hyaline cartilage and cancellous bone was observed in 5 of 16 defects. In contrast, no obvious repair or only fibrotic tissue was observed in Ctrl 2 and Ctrl 3. The compressive moduli of repaired cartilage in Exp reached 80.27% of the normal amount at 6 months, with a high level of glycosaminoglycan (GAG) content (no statistical difference from normal). In Ctrl 1, the compressive moduli and GAG content were 62.69% and 78.03% of normal levels, respectively. More importantly, GFP-labeled cells were detected in the engineered cartilage and the repaired subchondral bone. These results strongly indicate that the implanted BMSCs can differentiate into either chondrocytes or osteoblasts and repair articular osteochondral defects by forming engineered cartilage and engineered bone.     

Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between bone formation and scaffold resorption

Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (muCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The muCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed muX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.     

Meniscal regeneration using tissue engineering with a scaffold derived from a rat meniscus and mesenchymal stromal cells derived from rat bone marrow

The purpose of this study was to regenerate a meniscus using a scaffold from a normal meniscus and mesenchymal stromal cells derived from bone marrow (BM-MSCs). Thirty Sprague-Dawley rat menisci were excised and freeze-thawed three times with liquid nitrogen to kill the original meniscal cells. Bone marrow was aspirated from enhanced green fluorescent protein transgenic Sprague-Dawley rats. BM-MSCs were isolated, cultured for 2 weeks, and 2 x 10(5) cells were then seeded onto the meniscal scaffolds. Using a fluorescent microscope and immunohistochemical staining, repopulation of enhanced green fluorescent protein positive cells was observed in the superficial zone of the scaffold after 1 week of culture, and then in the deep zone after 2 weeks. At 4 weeks, expression of extracellular matrices was detected histologically and expression of mRNA for aggrecan and type X collagen was detected. Stiffness of the cultured tissue, assessed by the indentation stiffness test, had increased significantly after 2 weeks in culture, and approximated the stiffness of a normal meniscus. From this study, we conclude that a scaffold derived from a normal meniscus seeded with BM-MSCs can form a meniscus approximating a normal meniscus.     

冻存骨髓基质细胞复合材料体内成骨基质的合成能力

背景:课题组以往研究显示：体外培养条件下,冻存骨髓基质细胞复苏后仍保持较高的细胞存活率、细胞增殖及成骨分化能力。上述结果仍然需要进一步在体内环境下证实。 目的：观察经超低温冻存后的骨髓基质细胞和支架材料胶原膜BME-10X复合体植入裸鼠体内后Ⅰ型胶原的合成情况。 方法：体外分离培养Beagle犬骨髓基质细胞,冻存12个月后复苏,体外构建骨髓基质细胞和胶原膜材料复合体。分别经矿化诱导培养液、基础培养液培养5 d后,植入裸鼠体内,于术后第4周取出标本,进行大体观察、组织病理学和免疫组化分析,并应用图像分析系统对各组标本中的Ⅰ型胶原进行定量分析。以矿化诱导培养液培养的单纯胶原膜材料为对照组。 结果与结论：对照组在植入胶原膜后,胶原膜边界清晰,膜边缘及内部基本没有细胞生长,Ⅰ型胶原分布很少;在未诱导矿化组,术后第4周可见,胶原膜内有细胞长入,并有细小的条索状新生胶原形成,Ⅰ型胶原分布明显增多;在诱导矿化组,植入后也可见支架材料的分解降解和更多的细胞生长,大量新生的胶原形成类骨质样组织,与前两组对比,Ⅰ型胶原分布增多有显著性意义。结果表明冻存骨髓基质细胞复苏后进行体外培养扩增与诱导分化,并在体内环境下复合胶原支架材料,仍然具有较强成骨能力。     

Knitted poly-lactide-co-glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit Achilles tendon

The objectives of this study were to evaluate the morphology and biomechanical function of Achilles tendons regenerated using knitted poly-lactide-co-glycolide (PLGA) loaded with bone marrow stromal cells (bMSCs). The animal model used was that of an adult female New Zealand White rabbit with a 10-mm gap defect of the Achilles tendon. In group I, 19 hind legs with the created defects were treated with allogeneic bMSCs seeded on knitted PLGA scaffold. In group II, the Achilles tendon defects in 19 hind legs were repaired using the knitted PLGA scaffold alone, and in group III, 6 hind legs were used as normal control. The tendon-implant constructs of groups I and II were evaluated postoperatively at 2, 4, 8, and 12 weeks using macroscopic, histological, and immunohistochemical techniques. In addition, specimens from group I (n = 7), group II (n = 7), and group III (n = 6) were harvested for biomechanical test 12 weeks after surgery. Postoperatively, at 2 and 4 weeks, the histology of group I specimens exhibited a higher rate of tissue formation and remodeling as compared with group II, whereas at 8 and 12 weeks postoperation, the histology of both group I and group II was similar to that of native tendon tissue. The wound sites of group I healed well and there was no apparent lymphocyte infiltration. Immunohistochemical analysis showed that the regenerated tendons were composed of collagen types I and type III fibers. The tensile stiffness and modulus of group I were 87 and 62.6% of normal tendon, respectively, whereas those of group II were about 56.4 and 52.9% of normal tendon, respectively. These results suggest that the knitted PLGA biodegradable scaffold loaded with allogeneic bone marrow stromal cells has the potential to regenerate and repair gap defect of Achilles tendon and to effectively restore structure and function.     

Axon growth-promoting properties of human bone marrow mesenchymal stromal cells

Mesenchymal stromal cells are promising candidate donor cells for promoting functional tissue repair following traumatic spinal cord injury (SCI), however, the mechanism(s) of action remain poorly defined. Here, we describe an in vitro study of the axon growth-promoting properties of highly enriched populations of adult human mesenchymal stromal cells (hMSC). A random, non-oriented pattern of neuritic outgrowth was observed from dissociated adult rat DRG neurons seeded onto confluent A431 cells and PLL/laminin positive control substrata. Confluent hMSC formed arrays of similarly orientated cell bodies and processes which supported the regeneration of significantly more primary neurites but a slightly lower overall neuritic length than was observed over the PLL/laminin control substrate. The hMSC exerted a strong influence on the direction of neuritic outgrowth, with many regenerating processes following the orientation of underlying hMSC. The production of extracellular matrix appeared to be responsible for neuritic directionality, but the release of growth factors was a significant promoter for DRG neuritic outgrowth. This suggests that further investigations into the properties of hMSC may be of particular interest in the development of transplant-mediated strategies intending to promote functional axonal regeneration after SCI.     

Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors

Human bone marrow contains a population of bone marrow stromal cells (hBMSCs) capable of forming several types of mesenchymal tissues, including bone and cartilage. The present study was designed to test whether large cartilaginous and bone-like tissue constructs can be selectively engineered using the same cell population (hBMSCs), the same scaffold type (porous silk) and same hydrodynamic environment (construct settling in rotating bioreactors), by varying the medium composition (chondrogenic vs. osteogenic differentiation factors). The hBMSCs were harvested, expanded and characterized with respect to their differentiation potential and population distribution. Passage two cells were seeded on scaffolds and cultured for 5 weeks in bioreactors using osteogenic, chondrogenic or control medium. The three media yielded constructs with comparable wet weights and compressive moduli (25 kPa). Chondrogenic medium yielded constructs with higher amounts of DNA (1.5-fold) and glycosaminoglycans (GAG, 4-fold) per unit wet weight (ww) than control medium. In contrast, osteogenic medium yielded constructs with higher dry weight (1.6-fold), alkaline phosphatase (AP) activity (8-fold) and calcium content (100-fold) per unit ww than control medium. Chondrogenic medium yielded constructs that were weakly positive for GAG by contrast-enhanced MRI and alcian blue stain, whereas osteogenic medium yielded constructs that were highly mineralized by μCT and von Kossa stain. Engineered bone constructs were large (8 mm diameter × 2 mm thick disks) and resembled trabecular bone with respect to structure and mineralized tissue volume fraction (12%).     

透明质酸钠可作为成骨诱导后骨髓间充质干细胞的载体

目的:研究透明质酸钠作为组织工程骨支架的可行性.方法:体外培养兔骨髓间充质干细胞(MSCs),在成骨诱导剂地塞米松等的诱导下,向成骨细胞转化,并使之与透明质酸钠凝胶复合,通过倒置相差显微镜和扫描电子显微镜观察细胞贴附情况.结果:地塞米松等诱导组细胞形态向类成骨细胞转化,碱性磷酸酶表达明显增高,并表达Ⅰ型胶原.体外复合培养10 h,成骨细胞即开始于透明质酸钠凝胶中伸展生长,复合培养7 d,成骨细胞在凝胶中分化增殖,分泌细胞外基质.结论:适当浓度成骨诱导剂可成功地将兔MSCs向成骨细胞诱导,透明质酸钠是骨组织工程的良好载体.