Evaluation of human mesenchymal stem cells response to biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine composite scaffolds for bone tissue engineering

The aim of this study was to examine in vitro the response of human mesenchymal stem cells (hMSCs) on the novel biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine (BG-COL-HYA-PS) composite scaffold for potential use in bone tissue engineering. The initial attachment, the proliferation, migration and differentiation behavior of the cells on the BG-COL-HYA-PS composites were assessed in comparison with those on pure 58sBG, BG-COL, and BG-COL-HYA composites in either growth medium (L-DMEM supplemented with 10% fetal bovine serum) or osteogenic medium (growth medium supplemented with 0.1 microM dexamethasone, 10 mM beta-glycerophosphate, and 50 microM ascorbic acid). HMSCs attached, and subsequently proliferated and migrated on the BG-COL-HYA-PS composites to a significantly higher degree. The alkaline phosphatase (ALP) staining, ALP activity and the expression of the bone associated gene ALP, osteocalcin (OC), and osteopontin (OPN) was also significantly higher in the hMSCs on the BG-COL-HYA-PS scaffolds than those on the BG-COL, BG-COL-HYA composites and the pure 58sBG. These findings suggest that the BG-COL-HYA-PS composite porous scaffolds have high potential for use as scaffolds in bone tissue engineering and repair.     

Effect of strontium ions on the growth of ROS17/2.8 cells on porous calcium polyphosphate scaffolds

Preparation, characterization and cellular biocompatibility study of a series of calcium polyphosphate containing 0-100 mol% of Ca2+ replaced by Sr2+ were reported. The osteoblastic ROS17/2.8 cell line was used and seeded on the strontium-doped calcium polyphosphate (SCPP) scaffolds to estimate its optimal dose and to study its potential to support the growth of osteoblastic cells for bone tissue engineering. The effects of SCPP on cells' proliferation and differentiation were evaluated by MTT and ALP activity assay. The results showed that porous SCPP did not exert cytotoxic effect on the cells. In addition, the proliferation and differentiation of the growth of ROS17/2.8 cells on the SCPP containing a low dose of strontium showed a higher level compared to the control, and the SCPP containing 1% strontium was optimal according to the results of MTT and ALP activity assay. The cells on the porous SCPP formed a continuous layer on the outer and inner surface observed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The bunchy collagens were excreted from the cells and the calcium granules wrapped by collagens were sedimentated on the surface of cells. The results suggested that the biodegradable SCPP could stimulate the proliferation and differentiation of ROS17/2.8 cells in vitro after addition of proper dose of strontium. The porous SCPP may be a promising material for the bone tissue engineering.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

Solution-mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSCs) on PLGA by promoting the formation of a calcium-phosphate-rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells was tested. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSCs, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell-derived factors that promoted distant MSC differentiation. Thus PLGA-BG composites demonstrate significant potential as a bone-replacement material.     

Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.     

Calcium phosphate surface treatment of bioactive glass causes a delay in early osteogenic differentiation of adipose stem cells

Human adipose stem cells (ASCs) combined with osteostimulative material provide an attractive approach for clinical bone regeneration. The effect of calcium phosphate (Ca-P) surface treatment of three-dimensional bioactive glass scaffolds on the attachment, proliferation, and osteogenic differentiation of ASCs was studied. Three types of bioactive glass scaffolds (nontreated, thick and thin Ca-P treated) were compared. All scaffold types supported ASC attachment, spreading, and proliferation equally as detected by scanning electron microscopy, fluorescence staining, and DNA measurement. Indices of osteogenic differentiation including the expression of osteopontin and alkaline phosphatase (ALP) were consistently higher in the nontreated and thin Ca-P-treated scaffolds when compared with thick Ca-P-treated scaffolds at 2 weeks. ASCs cultured on nontreated bioactive glass scaffolds showed significantly higher ALP activity when compared with both thin and thick Ca-P-treated scaffolds after 1 week in culture, but these differences equalized between the three scaffolds by the 2-week time point. In conclusion, osteogenic differentiation appears to be delayed on the Ca-P surface-treated scaffolds. This delay is more pronounced with thick Ca-P treatment of the scaffolds.     

骨髓间充质干细胞与不同基质修饰的纳米晶胶原基骨体外生物相容性研究

研究大鼠骨髓间充质干细胞(MSCs)诱导培养后与不同基质修饰的纳米晶胶原基骨(nanoHydroxyapatite/collagen,nHAC)的生物相容性,为骨组织工程提供一种新型复合支架材料。SD大鼠MSCs经成骨诱导培养、扩增,进行成骨细胞表征后,种植与支架材料体外复合培养。实验分为4组:实验组A,纤维蛋白(FB)和纤维连接蛋白(FN)修饰的纳米晶胶原基骨((FB FN)-nHAC);实验组B,纤维蛋白修饰的纳米晶胶原基骨(FB-nHAC);实验组C,纤维连接蛋白修饰的纳米晶胶原基骨(FN-nHAC);对照组D,单纯的纳米晶胶原基骨(nHAC)。通过检测支架材料的细胞黏附率、不同时间点(3、7、10、14d)支架材料中细胞数、碱性磷酸酶活性以及扫描电镜观察细胞在材料上的生长状况,比较分析不同支架材料与细胞生物相容性差异。大鼠MSCs经诱导培养14d后,碱性磷酸酶细胞化学染色、I型胶原免疫荧光染色及矿化沉积茜素红染色均为阳性;细胞与支架材料黏附率A组最高为74.4%;支架材料中细胞数量均随培养时间延长而增长,且A组细胞数增加较快,与相同时相点其他各组材料中细胞数差异有显著性(P<0.05);各时相点细胞碱性磷酸酶活性表达A组最高,差异亦有显著性(P<0.05)。电镜观察发现4组材料上均有细胞生长,但A组的细胞生长状况明显好于其他组。大鼠MSCs经成骨诱导培养,可表达成骨细胞表型,(FB FN)-nHAC在体外实验中表现出与细胞优良的生物相容性,可作为较理想的新型复合支架应用于骨组织工程。     

Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells

In vitro culture techniques must be improved to increase the feasibility of cell-based tissue engineering strategies. To enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three-dimensional scaffolds. This system utilizes an elegant design to create a more effective environment for cell culture. In our design, hMSCs in the TPS bioreactor are encapsulated in alginate beads that are tightly packed in a tubular growth chamber. The medium is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds enhancing nutrient transfer while exposing the cells to shear stress. Results demonstrate that bioreactor culture supports early osteoblastic differentiation of hMSCs as shown by alkaline phosphatase gene expression. After 14 and 28 days of culture significant increases in the gene expression levels of osteocalcin, osteopontin, and bone morphogenetic protein-2 were observed with bioreactor culture, and expression of these markers was shown to increase with media flow rate. These results demonstrate the TPS bioreactor as an effective means to culture hMSCs and provide insight to the effect of long-term shear stresses on differentiating hMSCs.     

三维骨微管结构支架构造及体外培养研究

目的：三维骨微管结构支架构造方法研究以及成骨细胞复合后的体外培养，观察这种能够为细胞生长提供三维骨微管结构的支架对细胞贴附、生长、增殖以及分化的影响。方法：应用快速成形技术制造支架负型模具，在模具中填充CPC材料，待其固化后，去除模具，形成具有内部相互连通微管的三维支架。复合成骨细胞，进行体外培养。分别于第4d和14d取出样本，用扫描电镜观察细胞生长情况。结果：利用光固化快速成形技术间接构造所得三维支架，具有很好的三维立体结构。扫描电镜下观察，成骨细胞在三维支架表面和微管内贴附生长状况良好，并分泌大量基质。结论：三维骨微管结构支架的快速成形间接构造方法应用于骨组织工程中支架的构造是可行的，所构造的CPC支架结构能够使细胞在其表面和微管内生长、增殖和分化。     

Human bone cell cultures in biocompatibility testing. Part I: osteoblastic differentiation of serially passaged human bone marrow cells cultured in alpha-MEM and in DMEM

Well-characterised human osteoblastic bone marrow cell cultures are a useful in vitro tool to analyse bone tissue/biomaterials interactions. In this work, human bone marrow was cultured in experimental conditions described to favour osteoblastic differentiation and, serially passaged cells were cultured in two widely used culture media, minimum essential medium Eagle, alpha modification (alpha-MEM) and Dulbecco's modified Eagle's medium (DMEM). Cultures were grown for 35 d and compared concerning morphologic appearance on scanning electron microscopy (SEM), cell viability/proliferation, total protein content, activity of alkaline phosphatase (ALP) and ability to form calcium phosphate deposits. Results showed that cell proliferation was similar in cultures grown in the two media but ALP activity and ability to form mineralised deposits were lower in DMEM cultures. In both experimental situations, osteoblastic parameters were strongly reduced on cell passage, particularly from the first to the second subculture. In the experimental conditions used (presence of ascorbic acid, sodium beta-glycerophosphate and dexamethasone in the primary and secondary cultures), osteoblastic differentiation was observed in the first and second subcultures grown in alpha-MEM and in the first subculture grown in DMEM. These results underline the importance of the definition of the experimental conditions in studies involving bone cell cultures.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Insulin-like growth factor-I induces early osteoblast gene expression in human mesenchymal stem cells

Human adult mesenchymal stem cells (hMSCs) differentiate into an osteogenic lineage if the appropriate differentiative cues, such as dexamethasone or bone morphogenetic protein 2 (BMP-2), are present. This study was undertaken to determine the role of insulin-like growth factor I (IGFI) in the regulation of early osteoblast differentiation in hMSC. Previous studies have shown that IGF-I, regulates bone formation and remodeling by participating in the differentiation of mature cells of osteoblast lineage. We hypothesized that IGF-I exerted its effects early, but the effects were too subtle to be detected. Therefore, engineered hMSCs to produce IGF-I via adenoviral transfection and used quantitative real-time PCR (qPCR) to assess marker gene expression. Here we show that IGF-I up-regulates Type I collagen, Runx2, and alkaline phosphatase (Alp) gene expression in hMSCs, genes indicative of early osteogenic differentiation. We also observed mineral deposition in the absence of dexamethasone (Dex) in hMSC cultures treated with recombinant human BMP-2 after transduction with Ad-IGF-I. In conclusion Igf-I transduction up-regulated markers of osteoblastic differentiation and in conjunction with recombinant BMP-2-induced matrix mineralization independently of Dex (see Salasznyk et al., Stem Cells Dev 14(6):608-620, 2005, this issue).     

Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.     

The addition of biphasic calcium phosphate to porous chitosan scaffolds enhances bone tissue development in vitro

Uniform distribution of cells and their extracellular matrix is essential for the in vivo success of bone tissue engineering constructs produced in vitro. In this study, the effects of biphasic calcium phosphate (BCP) granules embedded into chitosan scaffolds on the distribution, morphology, and phenotypic expression of osteoblastic cells were investigated. Mesenchymal stem cells (MSCs) and preosteoblasts were cultured on chitosan scaffolds with and without BCP under osteoblastic differentiation/maturation conditions for periods up to 4 weeks. The addition of 25 wt % BCP to chitosan created a uniform layer of calcium phosphate (CaP) precipitation similar to bone mineral on the scaffold surfaces as determined by scanning electron microscopy and X-ray spectroscopy. Scaffolds with this CaP layer yielded more uniform and complete cell and ECM distribution than chitosan scaffolds without BCP. The suggestion of chemotaxis in the appearance of this response was confirmed by successive experiments in a Boyden chamber. The CaP layer also altered morphology of cells initially attached to the scaffold surfaces, leading to higher expression of marker proteins of osteoblastic phenotype including alkaline phosphatase and osteocalcin. The use of chitosan/BCP scaffolds for culture of MSCs and preosteoblasts enhances bone tissue development in vitro.     

In vitro cell performance on hydroxyapatite particles/poly(L-lactic acid) nanofibrous scaffolds with an excellent particle along nanofiber orientation

Highly porous hydroxyapatite (HA)/poly(L-lactide) (PLLA) nanofibrous scaffolds were prepared by incorporating needle-shaped nano- or micro-sized HA particles into PLLA nanofibers using electrospinning. The scaffolds had random or aligned fibrous assemblies and both types of HA particles were perfectly oriented along the fiber long axes. The biocompatibility and cell signaling properties of these scaffolds were evaluated by in vitro culture of rat osteosarcoma ROS17/2.8 cells on the scaffold surface. Cell morphology, viability and alkaline phosphatase (ALP) activity on each scaffold were examined at different time points. The HA/PLLA scaffolds exhibited higher cell viability and ALP activity than a pure PLLA scaffold. In addition, micro-sized HA particles supported cell proliferation and differentiation better than nano-sized ones in random scaffolds through a 10 day culture period and in aligned scaffolds at an early culture stage. The fibrous assembly of the scaffold had a pronounced impact on the morphology of the cells in direct contact with the scaffold surface, but not on cell proliferation and differentiation. Thus, HA/PLLA nanofibrous scaffolds could be good candidates for bone tissue engineering.     

Osteoblastic differentiation potential of human amniotic fluid-derived mesenchymal stem cells in different culture conditions

Osteoporosis is a bone degenerative disease characterized by a decrease in bone strength and an alteration in the osseous micro-architecture causing an increase in the risk of fractures. These diseases usually happen in post-menopausal women and elderly men. The most common treatment involves anti-resorptive agent drugs. However, the inhibition of bone resorption alone is not adequate for recovery in patients at the severe stage of osteoporosis who already have a fracture. Therefore, the combination of utilizing osteoblast micro mimetic scaffold in cultivation with the stimulation of osteoblastic differentiations to regain bone formation is a treatment strategy of considerable interest. The aims of this current study are to investigate the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid and to compare the monolayer culture and scaffold culture conditions. The results showed the morphology of cells in human amniotic fluid as f-type, which is a typical cell shape of mesenchymal stem cells. In addition, the proliferation rate of cells in human amniotic fluid reached the highest peak after 14 days of culturing. After which time, the growth rate slowly decreased. Moreover, the positive expression of specific mesenchymal cell surface markers including CD44, CD73, CD90, and also HLA-ABC (MHC class I) were recorded. On the other hand, the negative expressions of the endothelial stem cells markers (CD31), the hematopoietic stem cells markers (CD34, 45), the amniotic stem cells markers (CD117), and also the HLA-DR (MHC class II) were also recorded. The expressions of osteoblastogenic related genes including OCN, COL1A1, and ALP were higher in the osteogenic-induced group when compared to the control group. Interestingly, the osteoblastogenic related gene expressions that occurred under scaffold culture conditions were superior to the monolayer culture conditions. Additionally, higher ALP activity and greater calcium deposition were recorded in the extracellular matrix in the osteogenic-induced group than in the culture in the scaffold group. In summary, the mesenchymal stem cells derived from human amniotic fluid can be induced to be differentiated into osteoblastic-like cells and can promote osteoblastic differentiation using the applied scaffold.