Cells For Bone Tissue Engineering Research Articles

Evaluation of 45S5 Bioactive Glass Combined as a Bone Substitute in the Reconstruction of Critical Size Calvarial Defects in Rabbits

Biomaterial research and tissue engineering have guided new developments in bone replacement. In this study, the osteoconductive and osteoinductive properties of 45S5 Bioglass (Novabone-C/M, Porex Surg., Newnan, GA), granules as a bone replacement material for large calvarial defects were evaluated. Rabbit periosteal cells were expanded in culture and used in vivo. Alkaline-phosphatase assay, collagen type I, and calcium expression were applied to confirm osteoblast phenotype. In the in vivo phase, a 15-mm diameter critical size calvarial defect was created in rabbits (n = 14). The defect was reconstructed according to four treatment groups: autogenous bone (n = 2), Bioglass alone (n = 2), Bioglass + bone (n = 5), Bioglass + periosteal cells (n = 5). The animals were killed 12 weeks after surgery, and the samples were analyzed. Periosteal cells grew successfully in vitro. Because of their fast proliferation and potential to differentiate into osteoblasts, they were an excellent source of cells for bone tissue engineering. The best ossification was seen when autogenous bone was used (79.4% ossified), whereas only 8.2% of the defect in the Bioglass group showed ossification. Addition of bone or cells to the Bioglass increased the area of ossification to 42.7% and 30.2%, respectively. Defects replaced with Bioglass showed varying degrees of inflammatory reaction because of the intense cell-mediated biodegradation process. Based on these findings, the use of Bioglass granules to repair large craniofacial defects cannot be advised.

Adults Mesenchymal Stem Cells for Bone Tissue Engineering

Adults Mesenchymal Stem Cells for Bone Tissue Engineering

The role of muscle-derived stem cells in bone tissue engineering

The formation of bone and repair of bone defect requires a source of pluripotential mesenchymal stem cells. However, the capacity of the human body to generate bone components is limited. In this report, we show that the highly purified myogenic cells by the preplate technique have the capacity to differentiate into osteogenic lineage in vitro. The recombinant human bone morphogenic protein (rh-BMP-2) was immobilized on the molded gelatin composite. Primary muscle cells were isolated from newborn Wistar-rats calf muscle. The cells were then preplated in collagen-coated flasks. After six serial platings, the culture was enriched with small, round cells [pp6]. The effects of immobilized rhBMP-2 on the gelatin scaffold were evaluated by the analysis of alkaline phosphatase (ALP) and osteocalcin in culture medium after seedings of muscle-derived cells [pp6]. The results showed that the cells isolated from pp6 slow adhering cells possessed round mononuclear phenotype, marked ALP stain and matrix mineralization. The synthesis and secretion of ALP from pp6 muscle-derived cells were persistent higher than that of pp1–pp5 groups. The efficacy of rhBMP-2 immobilization on the gelatin scaffolds as manifested as the synthesis and secretion of ALP and osteocalcin from muscle-derived cells was always significantly higher than that of the control samples. In summary, our results suggest that the muscle-derived pp6 cells were capable of inducing and participating in bone formation. These results suggest that muscle tissue is a valuable resource for osteoprogenitor cells to be used in clinical practice to improve bone healing.

Osteogenesis characteristics of cultured rat mesenchymal stem cells under bone induction condition

To investigate the osteogenesis characteristics of cultured rat mesenchymal stem cells (MSCs) under bone induction condition. MSCs were isolated from adult rat by using density gradient separation method. The osteogenic inducers were compounds of Dexone, beta-glycerophosphate sodium and vitamin C. The MSC attachment formed soon after the seeding and grew into colonies with the appearance of fibroblastic cells. The osteogenic inducer with low dose of Dexone could promote the osteogenic differentiation of MSC. In the group of osteogenic inducer with low dose of Dexone, the expression of alkaline phosphatase (ALP) was remarkably increased after one week's induction, and the number of positive cells was (15.1 +/- 2.6), significantly higher than that of the control group (12.0 +/- 3.5) (P < 0.01). The calcified deposits began to appear in the group of osteogenic inducer with low dose of Dexone after one week's induction and was increased remarkably after three weeks, and the number of calcified deposits was (9.0 +/- 1.7), significantly higher than that of the control group (2.0 +/- 1.8) (P < 0.01). MSC can differentiate into osteogenesis by osteogenic induction and may be used to provide seed cells for bone tissue engineering.

Culture of the BMSCs of adult rhesus in vitro

目的 温对猴骨髓基质干细胞(BMSCs)进行体外培养及扩增，观察其原代及传代细胞的生长特点及生物学特点。方法 抽取4只成年猴髂骨骨髓，用全骨髓培养法进行体外培养获得BMSCs，胰酶消化传代，用条件培养基培养传代细胞。逐日倒置显微镜观察细胞生长情况，对传代细胞进行HE染色及碱性磷酸酶(ALP)染色。结果 成年雄性恒河猴BMSCs体外培养生长良好，原代细胞10-13d汇成单层，传代后4～7d长满瓶底。HE染色光镜下观察见BMSCs为单核细胞，细胞呈梭形、多角形，传代细胞碱性磷酸酶染色呈强阳性。结论 猴BMSc的体外培养增殖能力强，可诱导为成骨细胞，可作为灵长类动物骨组织工程的种子细胞。

Biocompatibility studies on fibrin glue cultured with bone marrow mesenchymal stem cells in vitro.

By culturing bone marrow mesenchymal stem cells of rabbits with fibrin glue in vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4-6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering.

Experimental study on human mesenchymal stem cells as seeding cells for bone tissue engineering

目的 从数量与功能两方面探讨人间充质干细胞 (human mesenchymal stem cells, hMSCs)作为骨组织工程种子细胞的可行性.方法髂骨穿刺,梯度离心分离获得 hMSCs,体外扩增培养;对第 3代细胞用地塞米松、β-甘油磷酸、维生素 C等成骨诱导培养,检测细胞碱性磷酸酶、骨钙素的表达和钙结节形成情况;诱导细胞与可吸收性复合支架材料体外构建复合体,植入裸小鼠皮下,对照组仅植入支架材料,术后 3周取材检测骨钙素、Ⅰ型胶原表达.结果 4ml骨髓含 3.2× 107～ 6.0× 107个单个核细胞,培养 3周收获 hMSCs 2.5× 107～ 4.3× 107;成骨诱导后,表达碱性磷酸酶、骨钙素阳性细胞数 >50%,对照组 < 10%;诱导细胞与材料复合后植入体内仍然高表达骨钙素、Ⅰ型胶原.结论 hMSCs能够满足体外构建组织工程化骨,对种子细胞数量和功能的要求.

Morphological observation of fetal bone mesenchymal stem cells cultured with PLGA scaffold

目的 探讨胎儿骨髓间充质干细胞( fetal bone marrow mesenchymal stem cells, fBMSCs)与低热高压法制作的聚丙交酯-乙交酯 (PLGA)支架材料复合培养,构建组织工程化骨的可行性. 方法预制 PLGA支架材料,取 5月胎儿肱骨和股骨骨髓,用单核细胞分离液分离 fBMSCs,含双抗的低糖 DMEM原代和传代培养,倒置显微镜下观察细胞生物学特性,用 FITC标记的抗 CD105、 CD44和用 PE标记的抗 CD34、 CD29对第 4代细胞进行流式细胞鉴定,并与 PLGA支架材料复合培养 1周后行扫描电镜观察. 结果原代及传代培养的 fBMSCs增殖较迅速,第 4代 CD105阳性细胞占 84.08%, CD29阳性细胞占 88.77%, CD44阳性细胞占 89.53%.复合 PLGA支架材料细胞生长状态良好. 结论骨髓间充质干细胞( bone mesenchymal stem cells,BMSCs)是骨组织工程适宜的种子细胞,与低热高压制作的 PLGA支架材料复合可体外构建组织工程骨.

Genetic marking with the ΔLNGFR-gene for tracing goat cells in bone tissue engineering

The use of bone marrow derived stromal cells (BMSC's) for bone tissue engineering has gained much attention as an alternative for autologous bone grafting. Little is known however, about the survival and differentiation of the cells, especially in the clinical application. The aim of this study was to develop a method to trace goat BMSC's in vivo. We investigated retroviral genetic marking, which allows stable expression of the label with cell division. Goat BMSC's were subjected to an amphotropic envelope containing a MoMuLV-based vector expressing the human low affinity nerve growth factor receptor (ΔLNGFR). Labeling efficiency and effect on the cells were analyzed. Furthermore, transduced cells were seeded onto porous ceramic scaffolds, implanted subcutaneously in nude mice and examined after successive implantation periods. Flow cytometry indicated a transduction efficiency of 40–60%. Immunohistochemistry showed survival and subsequent bone formation of the gene-marked cells in vivo. Besides, marked cells were also found in cartilage and fibrous tissue. These findings indicate the maintenance of the precursor phenotype following gene transfer as well as the ability of the gene to be expressed following differentiation. We conclude that retroviral gene marking with ΔLNGFR is applicable to trace goat BMSC's in bone tissue engineering research.

ALLOGENEIC MESENCHYMAL STEM CELLS REGENERATE BONE IN A CRITICAL-SIZED CANINE SEGMENTAL DEFECT

Mesenchymal stem cells from adult bone marrow are multipotent cells capable of forming bone, cartilage, and other connective tissues. In a previous study, we demonstrated that autologous mesenchymal stem cells could repair a critical-sized bone defect in the dog. The objective of this study was to determine whether the use of allogeneic mesenchymal stem cells could heal a critical-sized bone defect in the femoral diaphysis in dogs without the use of immunosuppressive therapy. A critical-sized segmental bone defect, 21 mm in length, was created in the mid-portion of the femoral diaphysis of twelve adult dogs that weighed between 22 and 25 kg. Each defect was treated with allogeneic mesenchymal stem cells loaded onto a hollow ceramic cylinder consisting of hydroxyapatite-tricalcium phosphate. A complete mismatch between donor stem cells and recipient dogs was identified by dog leukocyte antigen typing prior to implantation. The healing response was evaluated histologically and radiographically at four, eight, and sixteen weeks after implantation. The radiographic and histological results at sixteen weeks were compared with the historical data for the control defects, which included defects that had been treated with a cylinder loaded with autologous mesenchymal stem cells, defects treated with a cylinder without mesenchymal stem cells, and defects that had been left untreated (empty). The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells. For defects treated with allogeneic mesenchymal stem cell implants, no adverse host response could be detected at any time-point. Histologically, no lymphocytic infiltration occurred and no antibodies against allogeneic cells were detected. Histologically, by eight weeks, a callus spanned the length of the defect, and lamellar bone filled the pores of the implant at the host bone-implant interface. Fluorescently labeled allogeneic cells were also detected. At sixteen weeks, new bone had formed throughout the implant. These results were consistent with those seen in implants loaded with autologous cells. Implants loaded with allogeneic or autologous stem cells had significantly greater amounts of bone within the available pore space than did cell-free implants at sixteen weeks (p < 0.05). The results of this study demonstrated that allogeneic mesenchymal stem cells loaded on hydroxyapatite-tricalcium phosphate implants enhanced the repair of a critical-sized segmental defect in the canine femur without the use of immunosuppressive therapy. No adverse immune response was detected in this model.

Genetically modified mesodermal-derived cells for bone tissue engineering.

Alternatives to autografts and allografts for accelerated healing of skeletal defects. This article discusses the use of cells, isolated from tissues originating from the mesodermal germ layer and genetically modified to express bone morphogenetic protein, for bone tissue engineering applications.

Periosteal Cells in Bone Tissue Engineering

In 1742, H.L. Duhamel published a report in which the osteogenic function of periosteum was described. In 1932 H.B. Fell was the first to successfully culture periosteum; Fell concluded that this tissue might have the capability to form mineralized tissue in vitro. In the 1990s the research group of A.L. Caplan pioneered work exploring the osteogenic potential of periosteal cells in the field of bone engineering. On the basis of these studies a number of research groups have developed hard tissue generation concepts that aim to repeat the clinical success of bone autografts by culturing cells from periosteum and seeding a sufficient quantity of those cells into scaffolds made of biomaterials of natural and synthetic origin. The highly porous matrices support the induction of bone regeneration by creating and maintaining a space that facilitates progenitor cell migration, proliferation, and differentiation as well as graft revascularization. In this way, a host tissue-scaffold cell interphase might be created that allows reproduction of the intrinsic properties of autogenous bone, including the ability to be incorporated into the surrounding host bone and to continue normal bone-remodeling processes. This review discusses the history and state of the art of bone tissue engineering from a periosteum and periosteal cell source point of view and attempts to indicate future research directions.

Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells

The purpose of this research was to develop porous poly( D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbecco's phosphate buffered saline at 37°C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.

In vitro control of human bone marrow stromal cells for bone tissue engineering.

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.