Human osteoprogenitor bone formation using encapsulated bone morphogenetic protein 2 in porous polymer scaffolds

The ability to deliver, over time, biologically active osteogenic growth factors by means of designed scaffolds to sites of tissue regeneration offers tremendous therapeutic opportunities in a variety of musculoskeletal diseases. The aims of this study were to generate porous biodegradable scaffolds encapsulating an osteogenic protein, bone morphogenetic protein 2 (BMP-2), and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. BMP-2-encapsulated poly(DL-lactic acid) (PLA) scaffolds were generated by an innovative supercritical fluid process developed for solvent-sensitive and thermolabile growth factors. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on three-dimensional scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor-encapsulated scaffolds was observed after culture in an ex vivo model of bone formation developed on the basis of the chick chorioallantoic membrane model. BMP-2-encapsulated polymer scaffolds showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation and within diffusion chambers implanted into athymic mice as assessed by X-ray analysis and immunocytochemistry. The generation of three-dimensional biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculoskeletal tissue engineering.     

Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials.     

VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells

New strategies such as combined utilization of growth factors may provide a better treatment for difficult fractures. We have demonstrated enhanced angiogenesis and osteogenesis through the actions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) on the osteogenic differentiation of a cloned mouse osteoprogenitor cell in vitro and ectopic bone formation in vivo. Human VEGF and BMP-6 genes expressed together produced a significant increase in alkaline phosphatase activity, expression of the RunX2 and osteocalcin genes and mineralization. Microcomputed tomographic analysis of subcutaneous implants consisting of cells transfected with VEGF and BMP-6 cDNA and delivered on a 3D poly (lactic-co-glycolic acid) scaffold confirmed the additive effects between VEGF and BMP-6. Ectopic bone formation in the VEGF plus BMP-6 group was greatest compared to that in either VEGF or BMP-6 alone. This is the first study that demonstrates osteogenesis in vitro and in vivo through the additive effects of VEGF and BMP-6.     

Improvement of porous beta-TCP scaffolds with rhBMP-2 chitosan carrier film for bone tissue application

Ceramic materials are osteoconductive matrices extensively used in bone tissue engineering approaches. The performance of these types of biomaterials can be greatly enhanced by the incorporation of bioactive agents and materials. It is previously reported that chitosan is a biocompatible, biodegradable material that enhances bone formation. In the other hand, bone morphogenetic protein-2 (BMP-2) is a well-known osteoinductive factor. In this work we coated porous beta-tricalcium phosphate (beta-TCP) scaffolds with recombinant human BMP-2 (rhBMP-2) carrier chitosan films and studied how they could modify the ceramic physicochemical properties, cellular response, and in vivo bone generation. Initial beta-TCP disks with an average diameter of 5.78 mm, 2.9 mm thickness, and 53% porosity were coated with a chitosan film. These coating properties were studied by X-ray diffraction, Fourier transform-infrared analysis, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray analysis (EDX). Treatment modified the scaffold porous distribution and increased the average hardness. The biocompatibility did not seem to be altered. In addition, adhered C2C12 cells expressed alkaline phosphatase activity, related to cell differentiation toward osteogenic lineage, due to the incorporation of rhBMP-2. On the other hand, in vivo observations showed new bone formation 3 weeks after surgery, a much shorter time than control beta-TCP ceramics. These results suggest that developed coating improved porous beta-TCP scaffold for bone tissue applications and added osteoinductive properties.     

Response of murine bone marrow-derived mesenchymal stromal cells to dry-etched porous silicon scaffolds

Porous silicon shows great promise as a bio-interface material due to its large surface to volume ratio, its stability in aqueous solutions and to the ability to precisely regulate its pore characteristics. In the current study, porous silicon scaffolds were fabricated from single crystalline silicon wafers by a novel xenon difluoride dry etching technique. This simplified dry etch fabrication process allows selective formation of porous silicon using a standard photoresist as mask material and eliminates the post-formation drying step typically required for the wet etching techniques, thereby reducing the risk of damaging the newly formed porous silicon. The porous silicon scaffolds supported the growth of primary cultures of bone marrow derived mesenchymal stromal cells (MSC) plated at high density for up to 21 days in culture with no significant loss of viability, assessed using Alamar Blue. Scanning electron micrographs confirmed a dense lawn of cells at 9 days of culture and the presence of MSC within the pores of the porous silicon scaffolds.     

Plasticity of bone marrow-derived stem cells

Stem cell plasticity refers to the ability of adult stem cells to acquire mature phenotypes that are different from their tissue of origin. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into bone, cartilage, and fat. In this article, we review the literature that lends credibility to the theory that highly plastic BMCs have a role in maintenance and repair of nonhematopoietic tissue. We discuss the possible mechanisms by which this may occur. Also reviewed is the possibility that adult BMCs can change their gene expression profile after fusion with a mature cell, which has brought into question whether this stem cell plasticity is real.     

Fasudil联合骨髓源神经干细胞对EAE小鼠的神经保护作用

目的:探讨Rho激酶抑制剂法舒地尔(fasudil)联合骨髓源神经干细胞(bone marrow-derived neural stem cells,BM-NSCs)对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠的神经保护作用。方法:32只雌性C57BL/6小鼠(8~10周龄),用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)免疫,制备EAE,随机分为对照(dd H_2O)组、fasudil组、BM-NSCs组和fasudil+BM-NSCs组,并分别给予相应处理。免疫后检测小鼠临床症状和相关神经营养因子的表达,Image-Pro Plus 6.0软件进行阳性细胞计数,Graph Pad Prism 5软件统计分析。结果:与dd H_2O组比较,fasudil+BM-NSCs处理组明显延迟小鼠的平均起病时间,降低最高临床评分,并缓解EAE小鼠的临床症状;fasudil组、BM-NSCs组和fasudil+BM-NSCs组中脑源性神经营养因子、胶质细胞源性神经营养因子、神经生长因子、神经营养因子3和睫状神经营养因子阳性细胞数均有不同程度的增加,其中,fasudil+BM-NSCs组上述神经营养因子的表达明显多于dd H_2O组、fasudil组和BM-NSCs组(P0.01)。结论:Fasudil联合BM-NSCs通过协同和叠加效应促进神经营养因子表达,改善中枢神经系统微环境,发挥神经保护作用,从而缓解EAE的临床症状。     

Engraftment of bone marrow-derived epithelial cells

The long-held concept that transplanted bone marrow (BM)-derived cells contribute only to cells of the hematopoietic system was challenged by data from our laboratory showing that a single male BM-derived cell could not only reconstitute the hematopoietic system of an irradiated female recipient, but could also lead to the generation of mature BM-derived epithelial cells in the liver, lung, skin, and gastrointestinal tract. Careful costaining and single-cell analyses have been used to rule out false positive cells due to inadequate detection techniques in microscopy or cell overlay. Since this initial discovery, we have sought to understand the mechanisms underlying the formation of BM-derived epithelial cells, and to evaluate their therapeutic use for gene therapy and/or tissue regeneration. Several reports have shown that donor BM-derived cells, possibly macrophages, can fuse with existing host epithelial cells to form heterokaryons that express both donor and tissue-specific markers. While this is certainly true for murine tyrosinemia models, we have used a Cre-lox system to demonstrate that fusion is not a requirement for the generation of BM-derived epithelial cells and is likely not responsible for the BM-derived epithelial cells generated after standard BM transplantation. In a proof of principal experiment for potential gene therapy applications, we have shown that autologous BM-derived cells transfected with a transgene prior to BM transplantation are able to develop into mature type-II pneumocytes that express the transgene. We also discuss future research directions in the field and the therapeutic potential of BM-derived epithelia, including ongoing work to test whether combined cell and gene therapy can be used therapeutically in preclinical mouse models of human disease.     

Three-dimensional cultivation of human osteoblast-like cells on highly porous natural bone mineral

In this study, we investigated the growth and extracellular matrix synthesis of human osteoblast-like cells on highly porous natural bone mineral. Human bone cells were isolated from trabecular bone during routine iliac crest biopsies. Under conventional culture conditions, trabecular bone cells were able to assume the organization of a three-dimensional structure on a porous natural bone mineral (Bio-Oss(R) Block). Scanning electron microscopy examination after 6 weeks revealed multiple cell layers on the trabecular block. Transmission electron microscopy examination after 6 weeks revealed the accumulation of mature collagen fibrils in the intracellular and extracellular spaces, and showed multilayered, rough endoplasmic reticulum as well as mitochondria-rich cells surrounded by dense extracellular matrix. These morphological observations suggest that the cell layer may resemble the natural three-dimensional structure. Biochemical analysis revealed that the hydroxylysylpyridinoline, lysylpyridinoline, and hydroxyproline content of the cell layer increased in a time-dependent manner, whereas in monolayer culture without natural bone mineral, no measurable amounts of hydroxylysylpyridinoline or lysylpyridinoline, and a barely measurable amount of hydroxyproline, were noted. Mature collagen extracted by ethylenediaminetetraacetic acid-demineralization from the cell layer on natural bone mineral showed an identical electrophoretic pattern to that observed in human bone, as evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The present study demonstrated an excellent biocompatibility of the highly porous natural bone mineral in a three-dimensional bone cell culture system, and thus its potential for tissue-engineered growth of human bone.     

Polymethylmethacrylate particles inhibit osteoblastic differentiation of bone marrow osteoprogenitor cells

Aseptic implant loosening of total joint replacements often results from particle-mediated bone loss, which may be a combined effect of osteolysis and suppressed bone formation. Bone regeneration in the prosthetic bed depends on the activity of osteoblasts and their differentiation from osteoprogenitors in the bone marrow. This study investigated the effects of polymethylmethacrylate (PMMA) particles on the ability of bone marrow osteoprogenitors to differentiate into osteoblasts in vitro. Murine bone marrow cells challenged with PMMA particles on the first day of differentiation in osteogenic medium showed a dose-dependent decrease in osteoprogenitor proliferation, alkaline phosphatase expression, and mineralization. Undifferentiated bone marrow cells pretreated with PMMA particles in nonosteogenic medium for 5 days also showed a dose-dependent loss in osteogenic potential, which was sustained throughout subsequent growth in particle-free, osteogenic medium. Bone marrow cells challenged with PMMA particles after the fifth day of differentiation in osteogenic medium showed significant reductions in cellular proliferation, but not alkaline phosphatase expression and mineralization, indicating that bone marrow cells were most sensitive to particle treatment during the first 5 days of differentiation. This study demonstrated that PMMA particles inhibit osteoblastic differentiation of bone marrow osteoprogenitor cells, which may contribute to periprosthetic bone loss and implant failure.     

Constructing a tissue-engineered ureter using a decellularized matrix with cultured uroepithelial cells and bone marrow-derived mononuclear cells

This study investigated the efficacy of the ureteral decellularized matrix (UDM) as a scaffold material for a tissue-engineered ureter, and the effect of bone marrow-derived mononuclear cells (BM-MNC) on the neovascularization of the scaffold. Canine ureters were treated with deoxycholic acid to remove all cells. Uroepithelial cells (UEC) were obtained from canine bladders, cultured, and then seeded onto the inner surface of the UDM before transplantation into the subcutaneous space of nude mice or the omentum of nude rats. The cultured UECs began showing vacuolar degeneration 3 days after transplantation and gradually disappeared thereafter. To facilitate neovascularization in the implant, BM-MNCs were seeded around the UDM before transplantation. This facilitated the survival of the UECs, which formed three to five cellular layers after 14 days. The mean microvessel density was significantly increased in tissues seeded with BM-MNCs. However, cell-tracking experiments revealed that the increased number of capillaries in the experimental group was not due to the direct differentiation of transplanted endothelial progenitor cells. Our results demonstrate that the UDM is a useful scaffold for a tissue-engineered ureter, especially when seeded with BM-MNCs to enhance angiogenesis.     

豚鼠骨髓间充质干细胞与纳米羟基磷灰石体外构建组织工程人工听骨

背景：人工合成的纳米羟基磷灰石具有良好的生物活性和相容性,植入体内后能在短时间内与体内的软硬组织形成紧密结合,因此成为广泛应用的骨组织工程材料。 目的：观察豚鼠骨髓间充质干细胞与纳米羟基磷灰石复合多孔陶瓷材料体外复合培养的结合程度,并分析其构建组织工程人工听骨的可行性。 方法：采用细胞差速贴壁法分离培养豚鼠骨髓间充质干细胞,使用流式细胞仪检测CD29、CD45、CD44进行间充质干细胞表面标记物鉴定,并检测其骨细胞分化能力。将骨髓间充质干细胞与纳米羟基磷灰石复合多孔陶瓷共培养1,3,5,7,10 d,电镜观察骨髓间充质干细胞与此种支架材料的复合情况。 结果与结论：培养3 d后,可见大量骨髓间充质干细胞贴附在材料表层,并且形态稳定,生长旺盛,增殖力强,具有极佳的延伸性;培养5 d后可见材料表面已全部覆盖骨髓间充质干细胞,细胞结合紧密,细胞表面可见大量分泌颗粒,胞体明显增大,边缘完整,呈纤维样伸长;7 d后细胞逐渐从材料表面脱落,仍附在材料表面的细胞出现“星形”改变,“伪足”增多;10 d后细胞呈片状脱落。说明纳米羟基磷灰石材料保持了它良好的生物相容性,利于细胞黏附、增殖,可结合大量的骨髓间充质干细胞,用于构建组织工程人工听骨。     

In vivo evaluation of a novel porous hydroxyapatite to sustain osteogenesis of transplanted bone marrow-derived osteoblastic cells

Biosynthetic bone grafts are considered to contain one or more of three critical components: osteoprogenitor cells, an osteoconductive matrix, and osteoinductive growth factors. The basic requirements of the scaffold material are biocompatibility, mechanical integrity, and osteoconductivity. A major design problem is satisfying these requirements with a single composite. In this study, we hypothesize that one composite that combines bone marrow-derived osteoblasts and a novel mechanical reinforced porous hydroxyapatite with good biocompatibility and osteoconductivity (HA/BMO) can reach these requirements. A novel sintered porous hydroxyapatite (HA) was prepared by the following procedures. The HA slurry was foamed by adding polyoxyethylenelaurylether (PEI) and mixing. The pores were fixed by crosslinking PEI with diepoxy compounds and the HA porous body was sintered at 1200 degrees C for 3 h. The HA sintered porous body had a high porosity (77%), and was completely interconnected. Average pore diameter was 500 microm and the interconnecting path 200 microm in diameter. The compressive (17 MPa) and three-point bending (7 MPa) strengths were high. For in vivo testing, the 2-week subcultured HA/BMO (+) composites were implanted into subcutaneous sites of syngeneic rats until 8 weeks after implantation. These implants were harvested at different time points and prepared for the biochemical analysis of alkaline phosphatase activity (ALP) and bone osteocalcin content (OCN), and histological analysis. ALP and OCN in the HA/BMO group were much higher than those in the HA without BMOs control group 1 week after implantation (p < 0.001). Light microscopy revealed mature bone formation in the HA/BMO composite 4 weeks after implantation. In the SEM study, mineralized collagenous extracellular matrix was noted in HA/BMO composite 2 weeks after implantation with numbers of active osteoblasts. We conclude that the composite of the novel HA and cultured BMOs has osteogenic ability in vivo. These results provide a basis for further studies on the use of this composite as an implant in orthopaedic surgery.     

Osteogenic differentiation and ectopic bone formation of canine bone marrow-derived mesenchymal stem cells in injectable thermo-responsive polymer hydrogel

This study describes an injectable, thermo-responsive hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) copolymer for bone tissue engineering. The wettability, temperature-dependent change of water content, and volume of HA-CPN hydrogel were measured, together with its biocompatibility in vitro and in vivo. The dried hydrogel morphology shows a three-dimensional, porous structure with interconnected pores. Canine bone marrow-derived mesenchymal stem cells (cBMSCs) were encapsulated in HA-CPN hydrogel and osteoinduction was assessed by comparing samples with different osteogenic differentiation induction times but with the same total cell culture time. Cell proliferation and time-dependent osteogenic differentiation, evident from secretion of extracellular matrix and formation of mineral deposits, were observed. The cells showed better proliferation in HA-CPN hydrogel than on tissue culture polystyrene after osteo-induced for 21 days and higher alkaline phosphatase activity regardless of osteo-induction times. Mineralization extent of cBMSCs in HA-CPN followed by Alizarin red stains showed positive stained nodules after osteo-induced longer than 7 days. The cells/hydrogel construct also showed increased mechanical strength and elasticity after osteogenic differentiation, and the increase could be correlated with osteo-induction time. In vivo studies confirmed the biocompatibility and bioresorption of the HA-CPN hydrogel and ectopic bone formation when the hydrogel was used as a cell carrier for osteo-induced cBMSCs and implanted in nude mice subcutaneously. Taken together, the results indicate the feasibility and efficacy of HA-CPN hydrogel as an injectable bone tissue engineering scaffold with cBMSCs.     

骨髓间充质干细胞联合维生素E对急性肾损伤炎症反应的抑制作用

 目的:探索骨髓间充质干细胞联合维生素E对急性肾损伤中炎症反应的抑制效果,开发针对急性肾损伤的新型治疗模式。方法:采用庆大霉素作为急性肾损伤药物,复制大鼠急性肾损伤模型,以骨髓间充质干细胞联合维生素E对急性肾损伤进行治疗,治疗结束后,收集大鼠的血浆和肾脏组织,采用实时荧光定量PCR及ELISA方法,评价血浆及组织中炎症蛋白的表达。结果:通过骨髓间充质干细胞联合维生素E对大鼠急性肾损伤模型进行治疗后,大鼠血浆中的炎症蛋白显著降低;在肾脏组织中,与单一应用骨髓间充质干细胞或维生素E相比,二者联合在某些炎症蛋白的抑制方面具有更明显的效果。结论: 骨髓间充质干细胞联合维生素E对急性肾损伤中炎症反应具有一定抑制效果,优于单一骨髓间充质干细胞或单一维生素E的治疗方式,所以,这种联合治疗模式在急性肾损伤的治疗方面更具有临床应用价值。     

The role of bone marrow-derived cells in fibrosis

There is a growing realization that bone marrow-derived cells (BMDCs) are a potential therapy for many diseases including ischemic heart disease, arterial stenosis and osteogenesis imperfecta. On the other hand, the fact that BMDCs may also contribute to fibrosis in many solid organs as well as to fibrosis surrounding tumours suggests that BMDCs are also involved in disease progression. This review focuses on the contribution of bone marrow cells to organ and tumour fibrosis, noting the utility of BMDCs as a potential new portal through which to direct anti-tumour therapies. Conversely, bone marrow cell therapy has been claimed to reduce fibrosis in some organs, highlighting a seemingly beneficial as opposed to a detrimental effect of BMDCs on organ fibrosis.     

beta-TCP microporosity decreases the viability and osteoblast differentiation of human bone marrow stromal cells

Association of osteoprogenitor cells to calcium phosphate ceramics is currently under intense investigation, for its considered ability to induce bone formation and therefore to allow the successful repair of large bone defects. However, if the first experimental and clinical studies provided promising results, lack of new bone formation has been reported in a large number of animal experiments. In this context and since it has been reported that in some conditions, calcium phosphate ceramic microstructure induces ectopic bone formation, we investigated the effects of ceramic microporosity on the behavior of osteoprogenitor cells for the development of hybrid materials. Human bone marrow stromal cells (BMSCs) were seeded on beta-tricalcium phosphate (TCP) ceramics with 0, 25, or 45% microporosity and cell adhesion, viability, and osteoblastic differentiation have been studied for three weeks. Cell counts, measurement of MTS conversion, and LDH activity indicated that microporosity decreased the viability of BMSCs in a time and rate-dependent manner. In addition, microporosity inhibited osteoblastic differentiation as compared with nonmicroporous ceramics, as revealed by decreased alkaline phosphatase activity and osteocalcin secretion. Results of this in vitro study therefore highlight a negative role for beta-TCP microporosity in the behavior of human osteoprogenitor cells.