Tissue‐engineered nanoclay‐based 3D in vitro breast cancer model for studying breast cancer metastasis to bone

Breast cancer (BrCa) preferentially spreads to bone and colonises within the bone marrow to cause bone metastases. To improve the outcome of patients with BrCa bone metastasis, we need to understand better the mechanisms underlying bone metastasis. Researchers have relied heavily upon in vivo xenografts due to limited availability of human bone metastasis samples. A significant limitation of these is that they do not have a human bone microenvironment. To address this issue, we have developed a nanoclay‐based 3D in vitro model of BrCa bone metastasis using human mesenchymal stem cells (MSCs) and human BrCa cells mimicking late stage of BrCa pathogenesis at the metastatic site. This 3D model can provide a microenvironment suitable for cell–cell and cell–matrix interactions whilst retaining the behaviour of BrCa cells with different metastasis potential (i.e., highly metastatic MDA‐MB‐231 and low metastatic MCF‐7) as shown by the production of alkaline phosphatase and matrix metalloproteinase‐9. The sequential culture of MSCs with MCF‐7 exhibited 3D tumouroids formation and also occurrence of mesenchymal to epithelial transition of cancer metastasis as evidenced by gene expression and immunocytochemistry. The unique and distinct behaviour of highly metastatic MDA‐MB‐231 and the low metastatic MCF‐7 was observed at the bone metastasis site. The changes to migratory capabilities and invasiveness in MDA‐MB‐231 in comparison with tumour growth with MCF‐7 was observed. Together, a novel bone‐mimetic 3D in vitro BrCa model has been developed that could be used to study mechanisms governing the later stage of cancer pathogenesis in bone.     

The tick‐derived inhibitor Ixolaris prevents tissue factor signaling on tumor cells

Summary Background: Tissue factor (TF) is frequently overexpressed in cancer cells and correlated with more aggressive tumor phenotypes and poor prognosis. In addition to promoting coagulation‐dependent metastasis and cancer‐associated thrombosis, tumor cell‐expressed TF mediates direct cell signaling involving the protease‐activated receptor (PAR) 2. Ixolaris is a tick‐derived inhibitor of the TF–factor (F)VIIa–Xa coagulation initiation complex which blocks primary tumor growth and angiogenesis in glioblastoma and melanoma models. Methods: In this study we address the anti‐tumor effects of Ixolaris in TF–VIIa–PAR2 signaling‐dependent breast cancer models, a xenograft model of highly aggressive human MDA‐MB‐231mfp cells and a syngeneic model of PAR2‐deficient and replete PyMT mouse mammary carcinoma cells. Results: Ixolaris potently inhibited the procoagulant activity of human MDA‐MB‐231mfp or murine PyMT breast cancer cells. Ixolaris blocked signaling by the ternary TF–FVIIa–FXa complex, and, surprisingly, at higher concentrations also the binary TF–FVIIa complex on MDA‐MB‐231 cells. We show that Ixolaris interacts with certain residues in the human VIIa protease domain that are involved in PAR2 cleavage. In contrast to human VIIa, Ixolaris was a poor inhibitor of murine TF–FVIIa signaling and did not attenuate PAR2‐dependent tumor growth in a syngeneic mouse model of breast cancer progression. Conclusion: These data show that Ixolaris inhibits PAR2 cleavage specifically by human TF signaling complexes and suggest that Ixolaris may block tumor growth of human cell models with ectopic FVIIa expression through inhibition of direct TF–FVIIa–PAR2 signaling as well as its anticoagulant activity.     

人三阴性乳腺癌细胞耐药株的建立及特性研究

目的建立人三阴性乳腺癌细胞株MDA-MB-231的多西紫杉醇(Doc)耐药模型(MDA-MB-231/Doc)和表阿霉素(Epi)耐药模型(MDA-MB-231/Epi),探讨其生物学特性。方法采用Doc和EPi低浓度逐步加量诱导法历时12个月分别建立MDA-MB-231/Doc和MDA-MB-231/Epi耐药细胞株。通过细胞形态学观察、MTT法和流式细胞术分析、比较其生物学特性,实时荧光定量PCR检测多药耐药基因(MDR1)mRNA表达,Western Blot法检测P糖蛋白(P-gp)、雌激素受体(ER)、孕激素受体(PR)和人表皮生长因子受体2(Her-2)的表达状况。结果所构建的MDA-MB-231/Doc和MDA-MB-231/Epi耐药株可分别在12nmol/L Doc和800nmol/L Epi中稳定生长,在相同的药物浓度下,耐药细胞株的生长增殖率明显高于亲代细胞,其耐药指数分别为亲代敏感细胞的8.32倍和64.93倍,且相互呈交叉耐药状态。与亲代细胞相比,两株耐药细胞处于G1期和G2期的细胞增加、处于S期的细胞减少,随撤药时间的延长,细胞的增殖速度加快。两株耐药株的MDR1基因表达水平增高,分别为亲代细胞的4.05倍和5.96倍,P-gp表达为阳性。与MCF-7细胞株相比,MDA-MB-231细胞株ER、PR、HER2表达阴性,是典型的三阴性乳腺癌细胞株。结论成功建立MDA-MB-231/Doc和MDA-MB-231/Epi的耐药细胞株,其生长及耐药性稳定。     

Hierarchical starch‐based fibrous scaffold for bone tissue engineering applications

Fibrous structures mimicking the morphology of the natural extracellular matrix are considered promising scaffolds for tissue engineering. This work aims to develop a novel hierarchical starch‐based scaffold. Such scaffolds were obtained by a combination of starch–polycaprolactone micro‐ and polycaprolactone nano‐motifs, respectively produced by rapid prototyping (RP) and electrospinning techniques. Scanning electron microscopy (SEM) and micro‐computed tomography analysis showed the successful fabrication of a multilayer scaffold composed of parallel aligned microfibres in a grid‐like arrangement, intercalated by a mesh‐like structure with randomly distributed nanofibres (NFM). Human osteoblast‐like cells were dynamically seeded on the scaffolds, using spinner flasks, and cultured for 7 days under static conditions. SEM analysis showed predominant cell attachment and spreading on the nanofibre meshes, which enhanced cell retention at the bulk of the composed/hierarchical scaffolds. A significant increment in cell proliferation and osteoblastic activity, assessed by alkaline phosphatase quantification, was observed on the hierarchical fibrous scaffolds. These results support our hypothesis that the integration of nanoscale fibres into 3D rapid prototype scaffolds substantially improves their biological performance in bone tissue‐engineering strategies. Copyright © 2008 John Wiley & Sons, Ltd.     

A collagen network phase improves cell seeding of open‐pore structure scaffolds under perfusion

Scaffolds with open‐pore morphologies offer several advantages in cell‐based tissue engineering, but their use is limited by a low cell‐seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open‐pore architecture of polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds increases human bone marrow stromal cells (hBMSCs) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL–TCP scaffolds, rapid prototyped with a honeycomb‐like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen‐free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSCs directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSCs under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5‐fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non‐crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell‐based therapies, collagen‐filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion‐based bioreactor devices to streamline graft manufacture. Copyright © 2011 John Wiley & Sons, Ltd.     

Rat bone marrow stromal cells‐seeded porous gelatin/tricalcium phosphate/oligomeric proanthocyanidins composite scaffold for bone repair

Repair of bone defects remains a major challenge in orthopaedic surgery. Bone tissue engineering is an attractive approach for treating bone loss in various shapes and amounts. The aim of this study was to prepare and evaluate the feasibility of a porous scaffold, which was composed of oligomeric proanthocyanidin crosslinked gelatin mixed with β‐tricalcium phosphate (GTP) and was seeded with bone marrow stromal cells (BMSCs) as a bone substitute. GTP scaffolds were made porous using a salt‐leaching method. The physicochemical properties of the scaffold were evaluated to determine the optimal salt:composite weight ratio. The results indicated that the GTP scaffold had a favourable macroporous structure and higher porosity when the salt:composite weight ratio was 4:1. Cytotoxic tests demonstrated that extracts from the GTP scaffolds promoted the proliferation of BMSCs. Rat BMSCs were seeded on a GTP scaffold and cultured in a spinner flask. After 2 weeks of culture, scanning electron microscopy observation showed that the cells adhered well to the surfaces of the pores in the scaffold. Moreover, this study explored the biological response of rat calvarial bone to the scaffold to evaluate its potential in bone tissue engineering. Bone defects were filled with BMSC‐seeded GTP scaffold and acellular GTP scaffold. After 8 weeks, the scaffold induced new bone formation at a bone defect, as was confirmed by X‐ray microradiography and histology. The BMSC‐seeded scaffold induced more new bone formation than did an acellular scaffold. These observations suggest that the BMSCs‐seeded GTP scaffold can promote the regeneration of defective bone tissue. Copyright © 2012 John Wiley & Sons, Ltd.     

Bioactive nano‐fibrous scaffold for vascularized craniofacial bone regeneration

There has been a growing demand for bone grafts for correction of bone defects in complicated fractures or tumours in the craniofacial region. Soft flexible membrane like material that could be inserted into defect by less invasive approaches; promote osteoconductivity and act as a barrier to soft tissue in growth while promoting bone formation is an attractive option for this region. Electrospinning has recently emerged as one of the most promising techniques for fabrication of extracellular matrix such as nano‐fibrous scaffolds that can serve as a template for bone formation. To overcome the limitation of cell penetration of electrospun scaffolds and improve on its osteoconductive nature, in this study, we fabricated a novel electrospun composite scaffold of polyvinyl alcohol (PVA)‐poly (ε) caprolactone (PCL)‐Hydroxyapatite based bioceramic (HAB), namely, PVA‐PCL‐HAB. The scaffold prepared by dual electrospinning of PVA and PCL with HAB overcomes reduced cell attachment associated with hydrophobic PCL by combination with a hydrophilic PVA and the HAB can contribute to enhance osteoconductivity. We characterized the physicochemical and biocompatibility properties of the new scaffold material. Our results indicate PVA‐PCL‐HAB scaffolds support attachment and growth of stromal stem cells; [human bone marrow skeletal (mesenchymal) stem cells and dental pulp stem cells]. In addition, the scaffold supported in vitro osteogenic differentiation and in vivo vascularized bone formation. Thus, PVA‐PCL‐HAB scaffold is a suitable potential material for therapeutic bone regeneration in dentistry and orthopaedics.     

Co‐transplantation of Wharton's jelly mesenchymal stem cell‐derived osteoblasts with differentiated endothelial cells does not stimulate blood vessel and osteoid formation in nude mice models

A major challenge in bone tissue engineering is the lack of post‐implantation vascular growth into biomaterials. In the skeletal system, blood vessel growth appears to be coupled to osteogenesis—suggesting the existence of molecular crosstalk between endothelial cells (ECs) and osteoblastic cells. The present study (performed in two murine ectopic models) was designed to determine whether co‐transplantation of human Wharton's jelly mesenchymal stem cell‐derived osteoblasts (WJMSC‐OBs) and human differentiated ECs enhances bone regeneration and stimulates angiogenesis, relative to the seeding of WJMSC‐OBs alone. Human WJMSC‐OBs and human ECs were loaded into a silicate‐substituted calcium phosphate (SiCaP) scaffold and then ectopically implanted at subcutaneous or intramuscular sites in nude mice. At both subcutaneous and intramuscular implantation sites, we observed ectopic bone formation and osteoids composed of host cells when WJMSC‐OBs were seeded into the scaffold. However, the addition of ECs was associated with a lower level of osteogenesis, and we did not observe stimulation of blood vessel ingrowth. in vitro studies demonstrated that WJMSC‐OBs lost their ability to secrete vascular endothelial growth factor and stromal cell‐derived factor 1—including when ECs were present. In these two murine ectopic models, our cell‐matrix environment combination did not seem to be optimal for inducing vascularized bone reconstruction.     

Development of a synthetic tissue engineered three‐dimensional printed bioceramic‐based bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro

Over the last decade there have been increasing efforts to develop three‐dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica‐containing calcium alkali orthophosphate, using, first, a replica technique – the Schwartzwalder–Somers method – and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 10⁹ cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D‐printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 10⁹ cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.     

人骨唾液蛋白非融合荧光蛋白载体的构建及在乳腺癌细胞中的表达

背景人骨唾液蛋白基因在矿化组织以外的易发生骨转移的人乳腺癌中表达.临床观察显示骨转移处的乳腺癌细胞骨唾液蛋白的表达量要高于原发部位的乳腺癌细胞,因此骨唾液蛋白有可能与肿瘤特异性骨转移的关系密切.研究乳腺癌骨转移可为将来临床的预防和治疗提供新的药物靶点.目的建立骨唾液蛋白的稳定表达乳腺癌细胞系,观察骨唾液蛋白在乳腺癌骨转移的整个过程中的作用.设计对照实验.单位华南理工大学生物科学与工程学院,解放军广州军区广州总医院医学实验中心.材料实验于2003-11/2004-03在解放军广州军区广州总医院医学实验室完成.质粒、菌种和细胞pIRES2-EGFP载体质粒,E.Coli.Top10、含有人骨唾液蛋白基因全长的克隆载体pB-hBSP和发生特异性骨转移以及脑转移的人乳腺癌细胞株MDA-MB-231BO和MDA-MB-231BR.方法将人骨唾液蛋白基因通过聚合酶链式反应的方法从构建好的pB-hBSP载体中亚克隆出来,在其5'和3'端分别引入BglⅡ和Pst Ⅰ限制性酶切位点,定向克隆至真核表达载体pIRES2-EGFP,构建重组载体pIRES2-hBSP-EGFP.利用脂质体转染的方法将构建好的重组质粒转入特异性脑转移和骨转移的乳腺癌细胞株MDA-MB-231BR和MDA-MB-231BO中.主要观察指标pIRES2-hBSP-EGFP重组表达载体的构建.重组表达载体pIRES2-hBSP-EGFP转染乳腺癌细胞.结果①成功构建人骨唾液蛋白和绿色荧光蛋白非融合表达的真核表达载体pIRES2-hBSP-EGFP.②成功转染特异骨转移和脑转移的乳腺癌细胞株,可在荧光显微镜下观察到荧光蛋白标记,人骨唾液蛋白得到相应表达.结论真核表达载体pIRES2-hBSP-EGFP的构建及转染可为骨唾液蛋白在乳腺癌骨转移中的作用的体内、外研究奠定一定的基础.     

Reduction of BMP6‐induced bone formation by calcium phosphate in wild‐type compared with nude mice

Nude mice have been extensively used to investigate the potency of tissue engineering strategies for bone repair. However, the contribution of pro‐inflammatory and proregenerative stimuli of the host for the process of new bone formation and integration remains poorly understood. In this study, ectopic bone formation was investigated in nude (Nu) versus wild‐type (WT) mice. Calcium phosphate (CaP) scaffolds (CopiOs [Zimmer] and Bio‐Oss [Geistlich]) were loaded with different concentrations of rhBMP6 (40, 120, and 240 ng/mm³ rhBMP6) and implanted subcutaneously in Nu (BALB/c and NMR1) and WT (BALB/c and c57BL/6) mice. CaP scaffolds loaded with rhBMP6 did not form bone in WT mice. However, in Nu mice, 40 ng/mm³ rhBMP6 was sufficient to generate relevant volumes of new bone at 6 weeks after implantation. Looking into potential underlying mechanisms, TNF‐α blocking antibodies were injected intraperitoneally but could not restore bone formation. Also, mouse periosteal cells (mPDCs) seeded in CopiOs loaded with rhBMP6 did not significantly improve the outcome. Abrogation of bone formation was associated with dense cellular infiltration, in particular with the presence of CD3+ T‐lymphocytes. To probe a correlation between calcium ions and impaired bone formation in WT mice, type 1 collagen gels were loaded with rhBMP6 and calcium chloride and injected subcutaneously. These gels generated new bone in WT mice despite the increased percentage of CD3+ cells at Day 3 after implantation as compared with control gels. Overall, this study illustrated the negative effect of the inflammatory host response on the bone‐forming capacity of rhBMP6 coated on bioceramic scaffolds.     

Design,fabrication and in vitro evaluation of a novel polymer‐hydrogel hybrid scaffold for bone tissue engineering

The development of a bone mechanically‐compatible and osteoinductive scaffold is important for bone tissue engineering applications, particularly for the repair and regeneration of large area critically‐sized bone defects. Although previous studies with weight‐bearing scaffolds have shown promising results, there is a clear need to develop better osteoinductive strategies for effective scaffold‐based bone regeneration. In this study, we designed and fabricated a novel polymer‐hydrogel hybrid scaffold system in which a load‐bearing polymer matrix and a peptide hydrogel allowed for the synergistic combination of mechanical strength and great potential for osteoinductivity in a single scaffold. The hybrid scaffold system promoted increased pre‐osteoblastic cell proliferation. Further, we biotinylated human recombinant bone morphogenetic protein 2 (rhBMP2), and characterized the biotin addition and its effect on rhBMP2 biological activity. The biotinylated rhBMP2 was tethered to the hybrid scaffold using biotin‐streptavidin complexation. Controlled release studies demonstrated increased rhBMP2 retention with the tethered rhBMP2 hybrid scaffold group. In vitro evaluation of the hybrid scaffold was performed with rat bone marrow stromal cells and mouse pre‐osteoblast cell line MC3T3‐E1 cells. Gene expression of alkaline phosphatase (ALP), collagen I (Col I), osteopontin (OPN), bone sialoprotein (BSP), Runx‐2 and osteocalcin (OC) increased in MC3T3‐E1 cells seeded on the rhBMP2 tethered hybrid scaffolds over the untethered counterparts, demonstrating osteoinductive potential of the hybrid graft. These findings suggest the possibility of developing a novel polymer‐hydrogel hybrid system that is weight bearing and osteoinductive for effective bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Osteogenic potential of adipogenic predifferentiated human bone marrow‐derived multipotent stromal cells for bone tissue‐engineering

In the present study, we evaluated the benefits of an adipogenic predifferentiation, the pathway most closely related to osteoblastogenesis, on the pro‐osteogenic potential of human adult multipotent bone marrow stromal cells (hBMSCs), both in vitro and in vivo. Adipogenic differentiation of hBMSCs for 14 days resulted in a heterogeneous cell population from which the most adipogenic‐committed cells were eliminated by their lack of readhesion ability. Our results provided evidence that the select adherent adipogenic differentiated hBMSCs (sAD+ cells) express a gene profile characteristic of both adipogenic and osteogenic lineages. In vitro, when cultured in osteogenic medium, sAD+ differentiated along the osteogenic lineage faster than undifferentiated hBMSCs. In vivo, in an ectopic mouse model, sAD+ exhibited a significantly higher bone formation capability compared with undifferentiated hBMSCs. We sought, then, to investigate the underlying mechanisms responsible for such beneficial effects of adipogenic predifferentiation on bone formation and found that this outcome was not linked to a better cell survival post‐implantation. The secretome of sAD+ was both proangiogenic and chemoattractant, but its potential did not supersede the one of undifferentiated hBMSCs. However, using co‐culture systems, we observed that the sAD+ paracrine factors were pro‐osteogenic on undifferentiated hBMSCs. In conclusion, adipogenic priming endows hBMSCs with high osteogenic potential as well as pro‐osteogenic paracrine‐mediated activity. This preconditioning appears as a promising strategy for bone tissue engineering technology in order to improve the hBMSC osteogenic potency in vivo.     

Behaviour of human mesenchymal stem cells on chemically synthesized HA–PCL scaffolds for hard tissue regeneration

Our goal was to characterize the response of human mesenchymal stem cells (hMSCs) to a novel composite scaffold for bone tissue engineering. The hydroxyapatite–polycaprolactone (HA–PCL) composite scaffolds were prepared by a sol–gel method at room temperature and the scaffold morphology was investigated by scanning electron microscopy (SEM)/energy‐dispersive spectroscopy (EDS) to validate the synthesis process. The response of two different lines of hMSCs, bone‐marrow‐derived human mesenchymal stem cells (BMSCs) and dental pulp stem cells (DPSCs) in terms of cell proliferation and differentiation into the osteoblastic phenotype, was evaluated using Alamar blue assay, SEM, histology and alkaline phosphatase activity. Our results indicate that tissue engineering by means of composite HA–PCL scaffolds may represent a new therapeutic strategy to repair craniofacial bone defects. Copyright © 2013 John Wiley & Sons, Ltd.     

A tissue‐like construct of human bone marrow MSCs composite scaffold support in vivo ectopic bone formation

Biocompatible and osteoconductive cell–scaffold constructs comprise the first and most important step towards successful in vivo bone repair. This study reports on a new cell–scaffold construct composed of gelatin‐based hydrogel and ceramic (CaCO₃/β‐TCP) particles loaded with human MSCs producing a tissue‐like construct applied as a transplant for in vivo bone formation. Bone marrow‐derived human MSCs were cultured in osteogenic induction medium. 5 × 10⁵ (P₂) cells were loaded on a mixture of hydrogel microspheres and ceramic particles, cultured in a rotating dynamic culture for up to 3 weeks. Both hydrogel microspheres and ceramic particles coalesced together to form a tissue‐like construct, shown by histology to contain elongated spindle‐like cells forming the new tissue between the individual particles. Cell proliferation and cell viability were confirmed by Alamar blue assay and by staining with CFDA, respectively. FACS analysis conducted before loading the cells, and after formation of the construct, revealed that the profile of cell surface markers remained unchanged throughout the dynamic culture. The osteogenic potential of the cells composing the tissue‐like construct was further validated by subcutaneous transplants in athymic nude mice. After 8 weeks a substantial amount of new bone formation was observed in the cell‐construct transplants, whereas no bone formation was observed in transplants containing no cells. This new cell construct provides a system for in vivo bone transplants. It can be tailored for a specific size and shape as needed for various transplant sites and for all aspects of regenerative medicine and biomaterial science. Copyright © 2009 John Wiley & Sons, Ltd.     

Induction of bone formation in biphasic calcium phosphate scaffolds by bone morphogenetic protein‐2 and primary osteoblasts

Bone tissue engineering strategies mainly depend on porous scaffold materials. In this study, novel biphasic calcium phosphate (BCP) matrices were generated by 3D‐printing. High porosity was achieved by starch consolidation. This study aimed to characterise the porous BCP‐scaffold properties and interactions of osteogenic cells and growth factors under in vivo conditions. Five differently treated constructs were implanted subcutaneously in syngeneic rats: plain BCP constructs (group A), constructs pre‐treated with BMP‐2 (group B; 1.6 µg BMP‐2 per scaffold), seeded with primary osteoblasts (OB) (group C), seeded with OB and BMP‐2 (group D) and constructs seeded with OB and pre‐cultivated in a flow bioreactor for 6 weeks (group E). After 2, 4 and 6 weeks, specimens were explanted and subjected to histological and molecular biological analyses. Explanted scaffolds were invaded by fibrovascular tissue without significant foreign body reactions. Morphometric analysis demonstrated significantly increased bone formation in samples from group D (OB + BMP‐2) compared to all other groups. Samples from groups B‐E displayed significant mRNA expression of bone‐specific genes after 6 weeks. Pre‐cultivation in the flow bioreactor (group E) induced bone formation comparable with group B. In this study, differences in bone distribution between samples with BMP‐2 or osteoblasts could be observed. In conclusion, combination of osteoblasts and BMP‐2 synergistically enhanced bone formation in novel ceramic scaffolds. These results provide the basis for further experiments in orthotopic defect models with a focus on future applications in orthopaedic and reconstructive surgery. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparative study of the osteogenic ability of four different ceramic constructs in an ectopic large animal model

Tissue‐engineered constructs combining bone marrow mesenchymal stem cells with biodegradable osteoconductive scaffolds are very promising for repairing large segmental bone defects. Synchronizing and controlling the balance between scaffold‐material resorption and new bone tissue formation are crucial aspects for the success of bone tissue engineering. The purpose of the present study was to determine, and compare, the osteogenic potential of ceramic scaffolds with different resorbability. Four clinically relevant granular biomaterial scaffolds (specifically, Porites coral, Acropora coral, beta‐tricalcium phosphate and banked bone) with or without autologous bone marrow stromal cells were implanted in the ectopic, subcutaneous‐pouch sheep model. Scaffold material resorption and new bone formation were assessed eight weeks after implantation. New bone formation was only detected when the biomaterial constructs tested contained MSCs. New bone formation was higher in the Porites coral and Acropora coral than in either the beta‐tricalcium phosphate or the banked bone constructs; furthermore, there was a direct correlation between scaffold resorption and bone formation. The results of the present study provide evidence that, among the biomaterials tested, coral scaffolds containing MSCs promoted the best new bone formation in the present study. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid healing of a critical‐sized bone defect using a collagen‐hydroxyapatite scaffold to facilitate low dose,combinatorial growth factor delivery

The healing of large, critically sized bone defects remains an unmet clinical need in modern orthopaedic medicine. The tissue engineering field is increasingly using biomaterial scaffolds as 3D templates to guide the regenerative process, which can be further augmented via the incorporation of recombinant growth factors. Typically, this necessitates supraphysiological doses of growth factor to facilitate an adequate therapeutic response. Herein, we describe a cell‐free, biomaterial implant which is functionalised with a low dose, combinatorial growth factor therapy that is capable of rapidly regenerating vascularised bone tissue within a critical‐sized rodent calvarial defect. Specifically, we demonstrate that the dual delivery of the growth factors bone morphogenetic protein‐2 (osteogenic) and vascular endothelial growth factor (angiogenic) at a low dose (5 μg/scaffold) on an osteoconductive collagen‐hydroxyapatite scaffold is highly effective in healing these critical‐sized bone defects. The high affinity between the hydroxyapatite component of this biomimetic scaffold and the growth factors functions to sequester them locally at the defect site. Using this growth factor‐loaded scaffold, we show complete bridging of a critical‐sized calvarial defect in all specimens at a very early time point of 4 weeks, with a 28‐fold increase in new bone volume and seven‐fold increase in new bone area compared with a growth factor‐free scaffold. Overall, this study demonstrates that a collagen‐hydroxyapatite scaffold can be used to locally harness the synergistic relationship between osteogenic and angiogenic growth factors to rapidly regenerate bone tissue without the need for more complex controlled delivery vehicles or high total growth factor doses.     

Non‐rigid calcium phosphate cement containing hydrogel microbeads and absorbable fibres seeded with umbilical cord stem cells for bone engineering

The need for bone repair has increased as the population ages. Non‐rigid calcium phosphate scaffolds could provide compliance for micro‐motions within the tissues and yet have load‐supporting strength. The objectives of this study were to: (a) develop a non‐rigid calcium phosphate cement (CPC) with microbeads and fibre reinforcement; and (b) investigate human umbilical cord mesenchymal stem cell (hUCMSC) proliferation, osteodifferentiation and mineralization on non‐rigid CPC for the first time. Non‐rigid CPC was fabricated by adding extra tetracalcium phosphate in the traditional CPC and by incorporating chitosan, absorbable fibres and hydrogel microbeads. The non‐rigid CPC–microbead scaffold possessed a strain‐at‐failure of 10.7%, much higher than the traditional CPC's strain of 0.05% which is typical for brittle bioceramics. Flexural strength of non‐rigid CPC–microbead was 4‐fold that of rigid CPC–microbead scaffold, while work‐of‐fracture (toughness) was increased by 20‐fold. The strength of non‐rigid CPC–microbead–fibre scaffold matched that of cancellous bone. hUCMSCs on non‐rigid CPC proliferated from 100 cells/mm² at 1 day to 600 cells/mm² at 8 days. Alkaline phosphatase, osteocalcin and collagen gene expressions of hUCMSCs were greatly increased, and the cells synthesized bone minerals. hUCMSCs on non‐rigid CPC–microbead–fibre constructs had higher bone markers and more mineralization than those on rigid CPC controls. In conclusion, this study developed the first non‐rigid, in situ‐setting calcium phosphate–microbead–fibre scaffold with a strain‐at‐failure exceeding 10%. hUCMSCs showed excellent proliferation, osteodifferentiation and mineralization on non‐rigid CPC scaffold. The novel non‐rigid CPC‐hUCMSC construct with good strength, high strain‐at‐failure and toughness, as well as superior stem cell proliferation, osteodifferentiation and mineralization, is promising for load‐bearing bone regeneration applications. Copyright © 2012 John Wiley & Sons, Ltd.