Bone tissue engineering with porous hydroxyapatite ceramics

The main principle of bone tissue engineering strategy is to use an osteoconductive porous scaffold in combination with osteoinductive molecules or osteogenic cells. The requirements for a scaffold in bone regeneration are: (1) biocompatibility, (2) osteoconductivity, (3) interconnected porous structure, (4) appropriate mechanical strength, and (5) biodegradability. We recently developed a fully interconnected porous hydroxyapatite (IP-CHA) by adopting the “form-gel” technique. IP-CHA has a three-dimensional structure with spherical pores of uniform size that are interconnected by window-like holes; the material also demonstrated adequate compression strength. In animal experiments, IP-CHA showed superior osteoconduction, with the majority of pores filled with newly formed bone. The interconnected porous structure facilitates bone tissue engineering by allowing the introduction of bone cells, osteotropic agents, or vasculature into the pores. In this article, we review the accumulated data on bone tissue engineering using the novel scaffold, focusing especially on new techniques in combination with bone morphogenetic protein (BMP) or mesenchymal stem cells.     

Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media

Solution-mediated surface reactions occur for most calcium phosphate-based biomaterials and may influence cellular response. A reasonable extrapolation of such processes observed in vitro to in vivo performance requires a deep understanding of the underlying mechanisms. We therefore systematically investigated the nature of ion reactivity of calcium-deficient hydroxyapatite (CDHA) by exposing it for different periods of time to standard cell culture media of different chemical composition (DMEM and McCoy medium, with and without osteogenic supplements and serum proteins). Kinetic ion interaction studies of principal extracellular ions revealed non-linear sorption of Ca2? (～50% sorption) and K? (～8%) as well as acidification of all media during initial contact with CDHA (48h). Interestingly, inorganic phosphorus (P(i)) was sorbed from McCoy medium (～50%) or when using osteogenic media containing β-glycerophosphate, but not from DMEM medium. Non-linear sorption data could be perfectly described by pseudo-first-order and pseudo-second-order sorption models. At longer contact time (21 days), and with frequent renewal of culture medium, sorption of Ca2? remained constant throughout the experiment, while sorption of P(i) gradually decreased in McCoy medium. In great contrast, CDHA began to release P(i) slowly with time when using DMEM medium. Infrared spectra showed that CDHA exposed to culture media had a carbonated surface chemistry, suggesting that carbonate plays a key role in the ion reactivity of CDHA. Our data show that different compositions of the aqueous environment may provoke opposite ion reactivity of CDHA, and this must be carefully considered when evaluating the osteoinductive potential of the material.     

Bone marrow cell gene expression and tissue construct assembly using octacalcium phosphate microscaffolds

Calcium phosphates have been widely used in bone and soft tissue applications and are of considerable interest as scaffold materials due to properties of osteoconduction, resorbability and in some cases osteoinduction. These materials are microcrystalline and as such are processed using sintering, surface coating or cement technologies. However calcium phosphates containing HPO(4)(2-) ions often have layered crystal structures and can form macrocrystals in an aqueous environment at room temperature and pressure. This study aimed to investigate the potential of octacalcium phosphate (OCP) crystals for the attachment, proliferation and differentiation of bone marrow stromal cells and the potential of these cell seeded crystals as 'building blocks' for manufacture of self-supporting macroscale tissue constructs. An inverse relationship between cell number and crystal surface area was found and marrow cells grown on OCP crystals expressed osteocalcin and osteopontin mRNA, markers of osteoblastic differentiation, even in the absence of inductive media additives. Self-supporting crystal tissue macroscale constructs could be fabricated by culturing cell loaded crystals in moulds of the desired shape. Due to the low packing efficiency as a consequence of the high aspect ratio of OCP crystals, this microscaffold approach may offer the potential for ex vivo construction of large volumes of tissue which forms as a physiologically vascularised tissue.     

Cell selective chitosan microparticles as injectable cell carriers for tissue regeneration

The detection, isolation and sorting of cells holds an important role in cell therapy and regenerative medicine. Also, injectable systems have been explored for tissue regeneration in vivo, because it allows repairing complex shaped tissue defects through minimally invasive surgical procedures. Here we report the development of chitosan microparticles with a size of 115.8 μm able to capture and expand a specific cell type that can also be regarded as an injectable biomaterial. Monoclonal antibodies against cell surface antigens specific to endothelial cells and stem cells were immobilized on the surface of the microparticles. Experimental results showed that particles bioconjugated with specific antibodies provide suitable surfaces to capture a target cell type and subsequent expansion of the captured cells. Primarily designed for an application in tissue engineering, three main challenges are accomplished with the herein presented microparticles: separation, scale-up expansion of specific cell type and successful use as an injectable system to form small tissue constructs in situ.     

慢病毒介导绿色荧光蛋白转染人胚胎干细胞及其培养

目的 稳定培养人胚胎干细胞,并通过慢病毒载体对其进行绿色荧光蛋白标记.方法 利用小鼠胚胎成纤维细胞作为饲养层或Matrigel作为基质培养人胚胎干细胞,包装带有GFP序列的慢病毒转染人胚胎干细胞.对转染前后的人胚胎干细胞进行了碱性磷酸酶和SSEA-3免疫组化鉴定.结果 在MEF饲养层和Matrigel上均可培养出呈克隆样生长,表达标志抗原的人胚胎干细胞,经慢病毒转染及抗生素筛选后仍可稳定表达GFP.结论 成功地培养了人胚胎干细胞系,并进行了GFP标记.     

Three-dimensional cell culture and tissue engineering in a T-CUP (tissue culture under perfusion)

The aim of this study was to develop and validate a simple and compact bioreactor system for perfusion cell seeding and culture through 3-dimensional porous scaffolds. The developed Tissue Culture Under Perfusion (T-CUP) bioreactor is based on the concept of controlled and confined alternating motion of scaffolds through a cell suspension or culture medium, as opposed to pumping of the fluid through the scaffolds. Via the T-CUP, articular chondrocytes and bone marrow stromal cells could be seeded into porous scaffolds of different compositions and architectures (chronOS, Hyaff-11, and Polyactive) at high efficiency (greater than 75%), uniformity (cells were well distributed throughout the scaffold pores), and viability (greater than 97%). Culture of articular chondrocytes seeded into 4-mm thick Polyactive scaffolds for 2 weeks in the T-CUP resulted in uniform deposition of cartilaginous matrix. Cultivation of freshly isolated human bone marrow nucleated cells seeded into ENGipore ceramic scaffolds for 19 days in the T-CUP resulted in stromal cell-populated constructs capable of inducing ectopic bone formation in nude mice. The T-CUP bioreactor represents an innovative approach to simple, efficient, and reliable 3D cell culture, and could be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system in the context of regenerative medicine.     

Myogenesis in hemopoietic tissue mesenchymal stem cell culture

The myogenic differentiation capacity of prenatal mesenchymal stem cells from the main sites of hemopoiesis (bone marrow, thymus, liver, and spleen) was studied. Myogenesis was observed in all studied cell cultures except splenic mesenchymal stem cells. Differentiating cells from the thymus, bone marrow, and liver were positively stained for skeletal muscle markers (myogenin and MyoD). Autonomously contracting structures positively stained for cardiotroponin I and slow muscle myosin, were detected in the same cultures. Our experiments revealed differences in differentiation of mesenchymal stem cells from hemopoietic organs depending on the source of cells. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 2, pp. 63–69, April, 2006     

Bone ingrowth in bFGF-coated hydroxyapatite ceramic implants

This experimental study was performed to evaluate angiogenesis, bone formation, and bone ingrowth in response to osteoinductive implants of bovine-derived hydroxyapatite (HA) ceramics either uncoated or coated with basic fibroblast growth factor (bFGF) in miniature pigs. A cylindrical bone defect was created in both femur condyles of 24 miniature pigs using a saline coated trephine. Sixteen of the 48 defects were filled with HA cylinders coated with 50 microg rhbFG, uncoated HA cylinders, and with autogenous transplants, respectively. Fluorochrome labelled histological analysis, histomorphometry, and scanning electron microscopy were performed to study angiogenesis, bone formation and bone ingrowth. Complete bone ingrowth into bFGF-coated HA implants and autografts was seen after 34 days compared to 80 days in the uncoated HA group. Active ring-shaped areas of fluorochrome labelled bone deposition with dynamic bone remodelling were found in all cylinders. New vessels could be found in all cylinders. Histomorphometric analysis showed no difference in bone ingrowth over time between autogenous transplants and bFGF-coated HA implants. The current experimental study revealed comparable results of bFGF-coated HA implants and autogenous grafts regarding angiogenesis, bone synthesis and bone ingrowth.     

Mass enucleation of tissue culture cell monolayers

Summary A method is described for cytochalasin B induced enucleation of cultured mammalian cells grown in monolayers and centrifuged in 75 cm² culture flasks. Enucleation yields of approximately 90% from normal human fibroblasts can be achieved routinely with a single centrifugation in the presence of 10 µg/ml cytochalasin B. No loss of cells due to flask breakage occurs at the maximum recommended rotor speed, and flasks can be successfully centrifuged several times.     

Rapid cell culture procedure for tissue samples

Cell culture is an integral part of genetic studies, including cytogenetic, metabolic, and DNA analyses. Standard culture procedures used today involve plating minced tissue explants in dishes and waiting 3 to 4 weeks for adequate growth. We describe a method that utilizes collagenase digestion of tissue biopsies, enabling cell harvest as soon as 3 days after culture initiation; this has been used successfully in our laboratory for cytogenetic and metabolic studies. Culture duration can be controlled by the cell plating density. Although collagenase digestion of tissues is commonly used in cytogenetics laboratories for tumor or chorionic villous dissociation, we have found that few labs have considered using this rapid and efficient procedure for routine tissue samples.     

Immunocytochemistry controls using cell culture

A continuing problem in cytology laboratories is the lack of adequate control material for immunocytochemical testing. Usual control procedures involve testing paraffin-embedded control materials along with the patient specimens. These control materials are fundamentally unlike cytologic preparations. We have developed a method to make control preparations for immunocytochemical analysis using cultured anaplastic cells with known antigenic features from commercial sources. Cell lines included melanoma, rhabdomyosarcoma, T-cell leukemia, and squamous-cell carcinoma. Modified Saccomano and acetone fixation coupled with the cytospin technique enabled good-quality preparations. Cell lines were tested with antibodies for HMB-45, actin, leukocyte common antigen (LCA), and cytokeratin, with avidin-biotin immunoperoxidase and diaminobenzidine (DAB) as chromogens. Our final preparations were easily interpretable with excellent morphologic preservation of cellular detail. Cultured cells provide a superior method for preparing almost unlimited numbers of control slides for immunocytochemistry for laboratories with access to a tissue culture facility. Diagn. Cytopathol. 17:74–79, 1997. © 1997 Wiley-Liss, Inc.     

Quantitation of ranaviruses in cell culture and tissue samples

A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV.     

组织和细胞体外培养现状

随着细胞生物学、分子生物学、生物工程等方面的进展,对细胞生存环境的了解不断增加,对细胞培养所需的成分和特殊因子等的了解不断增加,以及组织细胞培养技术的不断发展、完善,能成功在体外培养的细胞的种类越来越多.     

The behaviour of human ameloblastoma tissue in cell culture

Human ameloblastoma tissue was investigated using cell culture techniques, transmission electron microscopy and fluorescent microscopy. Cultured cellular morphology was dependent on the type of substratum, with polygonal cells predominant on collagen substrata in contrast to an elongated cellular morphology on glass substrata. The presence of tonofilaments and desmosomes confirmed the epithelial origin of these cells. The distribution of Con A surface receptors and cytoplasmic actin in the same cell was studied using a double fluorochrome technique. Incubation with fluorescein isothiocyanate-labelled Con A at 37°C for increasing time periods resulted in the Con A receptors showing progressive changes in staining patterns from clusters, to caps to perinuclear globules. Sequential changes in cytoplasmic actin, labelled by a specific anti-actin auto-antibody traced with rhodamine-labelled goat anti-human globulin, corresponded to the Con A staining patterns.     

In vitro investigation of titanium and hydroxyapatite dental implant surfaces using a rat bone marrow stromal cell culture system

In this study, rat bone marrow cells (RBM) were used to evaluate different titanium and hydroxyapatite dental implant surfaces. The implant surfaces investigated were: a titanium surface having a porous titanium plasma-sprayed coating (sample code Ti-TPS), a titanium surface with a deep profile structure (sample code Ti-DPS), an uncoated titanium substrate with a machined surface (sample code Ti-ma) and a machined titanium substrate with a porous hydroxyapatite plasma-sprayed coating (sample code Ti-HA). RBM cells were cultured on the disc-shaped test substrates for 14 days. The culture medium was changed daily and examined for calcium and phosphate concentrations. After 14 days specimens were examined by light microscopy, scanning electron microscopy, energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. All test substrates facilitated RBM growth of extracellular matrix formation. Ti-DPS and Ti-TPS to the highest degree, followed by Ti-ma and Ti-HA. Ti-DPS and Ti-TPS displayed the highest cell density and thus seem to be well suited for the endosseous portion of dental implants. RBM cells cultured on Ti-HA showed a delayed growth pattern. This may be related to its high phosphate ion release.     

Bone tissue engineering with human mesenchymal stem cell sheets constructed using magnetite nanoparticles and magnetic force

An in vitro reconstruction of three-dimensional (3D) tissues without the use of scaffolds may be an alternative strategy for tissue engineering. We have developed a novel tissue engineering strategy, termed magnetic force-based tissue engineering (Mag-TE), in which magnetite cationic liposomes (MCLs) with a positive charge at the liposomal surface, and magnetic force were used to construct 3D tissue without scaffolds. In this study, human mesenchymal stem cells (MSCs) magnetically labeled with MCLs were seeded onto an ultra-low attachment culture surface, and a magnet (4000 G) was placed on the reverse side. The MSCs formed multilayered sheet-like structures after a 24-h culture period. MSCs in the sheets constructed by Mag-TE maintained an in vitro ability to differentiate into osteoblasts, adipocytes, or chondrocytes after a 21-day culture period using each induction medium. Using an electromagnet, MSC sheets constructed by Mag-TE were harvested and transplanted into the bone defect in the crania of nude rats. Histological observation revealed that new bone surrounded by osteoblast-like cells was formed in the defect area 14 days after transplantation with MSC sheets, whereas no bone formation was observed in control rats without the transplant. These results indicated that Mag-TE could be used for the transplantation of MSC sheets using magnetite nanoparticles and magnetic force, providing novel methodology for bone tissue engineering.     

High yield tissue dispersing method for cell culture

Summary A reliable method for preparing primary cultures from different organs, using a mixture of trypsin and Versene as dispersing agent(s) is described. This method provides high cell yields as well as monolayers free of cellular debris and suitable for determining viral cytopathogenic effects.     

Bone formation-promoting effect of genistein on marrow mesenchymal cell culture

The effect of genistein, a soybean isoflavone, on new bone formation by bone marrow cells from mature rats and humans was examined. Bone marrow cells were collected from the femoral diaphysis of 7-week-old Fisher rats, cultured in MEM containing fetal calf serum and then cultured with or without the addition of dexamethasone to the bone-forming medium. Genistein was added at concentrations of 10(-5),10(-6),10(-7) or 10(-8) M. Bone formation was examined 2 weeks after culture. After informed consent was obtained from a 55-year-old woman with lumbar spondylosis deformans, bone marrow cells were collected from her ilium for culture by the same process, and bone formation investigated. In both rats and humans, when dexamethasone was added to the bone-forming medium, genistein (10(-7) M and 10(-8) M) caused a significant increase in the levels of calcium, alkaline phosphatase, and DNA compared with cells not cultured in genistein. In conclusion, genistein was found to promote bone formation at lower concentrations across species, and thus may be useful as a bone formation-promoting factor.     

Studies of renal cell function using cell culture techniques

Cell culture, a powerful tool for the study of cell biology, offers advantages for the study of renal cell function. Epithelial cells derived from a variety of organs, including the kidney, form oriented epithelial sheets in culture that have many structural characteristics (microvilli, tight junctions) of epithelia in situ. There is evidence of transepithelial transport of salt and water by cells of two lines (MDCK and LLC-PK1) derived from mammalian kidney. LLC-PK1 cells may also manifest the glucose transport system of the proximal tubule. Cells of both lines have adenylate cyclase activity sensitive to hormones. Two lines of cells derived from toad urinary bladder form epithelia with a high transepithelial resistance and transport sodium actively from apical to basolateral surface. The rate of sodium transport in both lines is stimulated by cyclic AMP and by aldosterone. There are important differences in the characteristics of the response of the two lines to aldosterone as well as in their sensitivity to inhibition of sodium transport by amiloride. These differences may lead to new insights regarding the molecular events in the response to aldosterone and in the inhibitory action of amiloride. Cultures of kidney cells have also been used effectively to study the biosynthesis of the hormonal derivative of vitamin D and to study prostaglandin production. In addition, cell culture is ideally suited for study of the developmental biology of the kidney.