Surface modification of a porous polyurethane through a culture of human osteoblasts and an electromagnetic bioreactor.

There is increasing interest in new biomaterials and new culture methods for bone tissue engineering, in order to produce, in vitro, living constructs able to integrate in the surrounding tissue. Using an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz), we investigated the effects of electromagnetic stimulation on SAOS-2 human osteoblasts seeded onto a porous polyurethane. In comparison with control conditions, the electromagnetic stimulation caused higher cell proliferation, increased surface coating with decorin and type-I collagen, and higher calcium deposition. The immunolocalization of decorin and type-I collagen showed their colocalization in the cell-rich areas. The use of an electromagnetic bioreactor aimed at obtaining the surface modification of the porous polyurethane in terms of cell colonization and coating with calcified matrix. The superficially modified biomaterial could be used, in clinical applications, as an implant for bone repair.     

Acceleration of bone formation with BMP2 in frame-reinforced carbonate apatite–collagen sponge scaffolds

The development is expected of scaffold biomaterials that feature a shape-maintaining property in addition to high porosity and large pores that cells can easily invade. To develop a new biodegradable scaffold biomaterial reinforced with a frame, synthesized carbonate apatite (CO₃Ap) was mixed with neutralized collagen gel, and the CO₃Ap–collagen mixtures were lyophilized into sponges in a porous hydroxyapatite (HAp) frame ring. X-ray diffraction and Fourier transform infrared spectroscopy (FT-IR) analyses together with chemical analysis indicated that the synthesized CO₃Ap had a crystalline nature and a chemical composition similar to that of bone. Scanning electron microscope (SEM) observation showed that the CO₃Ap–collagen sponge had a sui pore size for cell invasion. In proliferation and differentiation experiments with osteoblasts, alkaline phosphatase and osteopontin activity were clearly detected. When these sponge–frame complexes with bone morphogenic protein (rh-BMP2) were implanted beneath the periosteum cranii of rats, significant new bone was created at the surface of the periosteum cranii after 4 weeks of implantation. These reinforced CO₃Ap–collagen sponges with rh-BMP2 are expected to be used as hard tissue scaffold biomaterials for the therapeutic purpose of the rapid cure of bone defects.     

Calcified matrix production by SAOS-2 cells inside a polyurethane porous scaffold, using a perfusion bioreactor

The repair and regeneration of damaged or resected bone are problematic. Bone autografts show optimal skeletal incorporation, but often bring about complications. Hence, there is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic cross-linked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells within a porous scaffold. The polyurethane porous scaffold had an average pore diameter of 624 microm. Using a perfusion bioreactor, we investigated the effect of shear stress on SAOS-2 human osteoblast proliferation and calcified matrix production. The physical, morphological, and compressive properties of the polyurethane foam were characterized. At a scaffold perfusion rate of 3 mL/min, in comparison with static conditions without perfusion, we observed 33% higher cell proliferation; higher secretion of osteopontin, osteocalcin, decorin, and type I collagen (9.16-fold, 71.9-fold, 30.6-fold, and 18.12-fold, respectively); and 10-fold increased calcium deposition. The design of the bioreactor and the design of the polyurethane foam aimed at obtaining cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.     

Cell-surface interactions of rat tooth germ cells on various biomaterials

This is the first study to explore the effect of biomaterial on tooth germ cell adhesion and proliferation in vitro. The purpose of this study is to evaluate the effects of cell-surface interactions of tooth germ cells on biomaterials with various surface hydrophilicities. The biomaterials used in this study included polyvinyl alcohol (PVA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene-co-vinyl alcohol; EVAL), and polyvinylidene fluoride (PVDF). Cell morphology was observed by photomicroscopy. Cell growth was assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity and the characteristic expression of amelogenin and collagen type I in tooth germ cells was investigated using immunocytochemistry. The results indicated that adhesion and proliferation of tooth germ cells to biomaterials with moderate hydrophilicity/hydrophobicity was superior compared to most hydrophobic material PVDF or mosthydrophilic material PVA in this study. Cellular adhesion and proliferation was evident on all tested biomaterials except PVA. The cell spheroids on PVA appeared not to be proliferated and remained as well as reattachable to tissue culture plates. In conclusion, biomaterials with moderate hydrophilicity are suitable for adhesion and proliferation of tooth germ cells. The material PVA may be a good biomaterial for maintaining tooth germ cells in three-dimensional biological restoration.     

How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials

The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h 6 h. 12 h, 24 h) and the seeding concentration (1 x 10(5), 1 x 10(6), 2 x 10(6)cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 x 10(5) cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery.     

Bone regeneration performance of surface-treated porous titanium

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid–alkali (AcAl), alkali–acid–heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial.     

The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogel-polyHIPE polymer hybrid material

The objective of this study was to investigate the effect of combining two biomaterials on osteoblast proliferation, differentiation and mineralised matrix formation in vitro. The first biomaterial has a well-defined architecture and is known as PolyHIPE polymer (PHP). The second biomaterial is a biologically inspired self-assembling peptide hydrogel (RAD16-I, also called PuraMatrix) that produces a nanoscale environment similar to native extracellular matrix (ECM). Our work investigates the effect of combining RAD16-I with two types of PHP (HA (Hydroxyapatite)-PHP and H (Hydrophobic)-PHP) and evaluates effects on osteoblast growth and differentiation. Results demonstrated successful incorporation of RAD16-I into both types of PHP. Osteoblasts were observed to form multicellular layers on the combined biomaterial surface and also within the scaffold. Dynamic cell seeding and culturing techniques were compared to static seeding methods and produced a more even distribution of cells throughout the constructs. Cells were found to penetrate the scaffold to a maximum depth of 3 mm after 35 days in culture. There was a significant increase in cell number in H-PHP constructs coated with RAD16-I compared to H-PHP alone. Our results show that RAD16-I enhances osteoblast differentiation and indicates that the incorporation of this peptide provides a more permissive environment for osteoblast growth. We have developed a microcellular polymer containing a nanoscale environment to enhance cell: biomaterial interactions and promote osteoblast growth in vitro.     

Surface modification by complexes of vitronectin and growth factors for serum-free culture of human osteoblasts

Cell attachment, expansion, and migration in three-dimensional biomaterials are crucial steps for effective delivery of osteogenic cells into bone defects. Complexes composed of vitronectin (VN), insulin-like growth factors (IGFs), and insulin growth factor-binding proteins (IGFBPs) have been reported to enhance cell attachment, proliferation, and migration in a variety of cell lines in vitro. The aim of this study was to examine whether prebound complexes of VN and IGFs +/- IGFBPs could facilitate human osteoblast serum-free expansion in vitro and enhance cell attachment, proliferation, and migration in three-dimensional biomaterial constructs. Human osteoblasts derived from alveolar bone chips and the established human osteoblast cell line Saos-2 were used. These cells were seeded on tissue culture plates and porous scaffolds of type I collagen sponges and polyglycolic acid (PGA), which had been coated with VN +/- IGFBP-5 +/- IGF-I. Cell attachment, proliferation, and migration were evaluated by cell counting, confocal microscopy, and scanning electron microscopy. The number of attached human osteoblasts was significantly higher in VN-coated polystyrene culture dishes. Furthermore, significant increases in cell proliferation were observed when growth factors were bound to these surfaces in the presence of VN. In the two scaffold materials examined, greater cell attachment was found in type I collagen sponges compared with PGA scaffolds. However, coating the scaffolds with complexes composed of VN + IGF-I or VN + IGFBP-5 + IGF-I enhanced cell attachment on PGA. Moreover, the presence of VN + IGFBP-5 + IGF-I resulted in significantly greater osteoblast migration into deep pore areas as compared with untreated scaffolds or scaffolds treated with fetal calf serum. These results demonstrated that complexes of VN + IGFBP-5 + IGF-I can be used to expand osteoblasts in vitro under serum-free conditions and enhance the attachment and migration of human osteoblasts in three-dimensional culture. This in turn suggests a potential application in surface modification of biomaterials for tissue reconstruction.     

Ectopic bone formation in rat marrow stromal cell/titanium fiber mesh scaffold constructs: effect of initial cell phenotype

Titanium fiber mesh scaffolds have been shown to be a suitable material for culture of primary marrow stromal cells in an effort to create tissue engineered constructs for bone tissue replacement. In native bone tissue, these cells are known to attach to extracellular matrix molecules via integrin receptors for specific peptide sequences, and these attachments can be a source of cell signaling, affecting cell behaviors such as differentiation. In this study, we examined the ability of primary rat marrow stromal cells at two different stages of osteoblastic differentiation to further differentiate into osteoblasts both in vitro and in vivo when seeded on titanium fiber mesh scaffolds either with or without RGD peptide tethered to the surface. In vitro, the tethered RGD peptide resulted in reduced initial cell proliferation. In vivo, there was no effect of tethered RGD peptide on ectopic bone formation in a rat subcutaneous implant model. Scaffold/cell constructs exposed to dexamethasone for 4 days prior to implantation (+dex constructs) resulted in significant bone formation whereas no bone formation was observed in--dex constructs. These results show that the osteoblastic differentiation of marrow stromal cells was not dependent on surface tethered RGD peptide, and that the initial differentiation stage of implanted cells plays an important role in bone formation in titanium fiber mesh bone tissue engineering constructs.     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability,morphology, and function

Tissue engineering with three-dimensional biomaterials represents a promising approach for developing hepatic tissue to replace the function of a failing liver. Herein, we address cell seeding and distribution within porous alginate scaffolds, which represent a new type of porous biomaterial for tissue engineering. The hydrophilic nature of the alginate scaffold as well as its pore structure and interconnectivity enabled the efficient seeding of hepatocytes into the scaffolds, that is, 70-90% of the initial cells depending on the seeding method. Utilization of centrifugal force during seeding enhanced cell distribution in the porous scaffolds, consequently enabling the seeding of concentrated cell suspensions (>1 x 10(7) cells/mL). Cell density in scaffolds affected hepatocyte viability as judged by MTT assay. At a cell density of 0.28 x 10(6) cells/cm3 scaffold, the number of viable hepatocytes decreased to 33% of its initial value within 7 days, whereas at the denser cultures, 5.7 x 10(6) cells/cm3 scaffold and higher, the cells maintained higher viability while forming a network of connecting spheroids. In the high-density cellular constructs, hepatocellular functions such as albumin and urea secretion, and detoxification (cytochrome P-450 and phase II conjugating enzyme activities), remained high during the 7-day culture. Collectively, the results of the present study highlight the importance of cell density on the hepatocellular functions of three-dimensional hepatocyte constructs as well as the advantages of alginate matrices as scaffoldings.     

Effects of biomaterial-induced inflammation on fibrosis and rejection

Evidence is emerging that biomaterials cause inflammation by ligating innate immune receptors on antigen presenting cells. Although inflammation is usually viewed as detrimental, it has unexpected and potentially beneficial effects on fibrosis and transplant rejection. For example, the magnitude of inflammation due to a biomaterial is not predictive of the extent of fibrosis. Similarly, biomaterials do not always show adjuvancy. Some biomaterials suppressed T cell rejection responses in vivo and in vitro, while others non-specifically stimulated T cell proliferation. Understanding these complex inter-relationships is the key to designing a biomaterial that stimulates regeneration and induces tolerance in tissue engineering applications.     

The Advancement of Biomaterials in Regulating Stem Cell Fate

Stem cells are well-known to have prominent roles in tissue engineering applications. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can differentiate into every cell type in the body while adult stem cells such as mesenchymal stem cells (MSCs) can be isolated from various sources. Nevertheless, an utmost limitation in harnessing stem cells for tissue engineering is the supply of cells. The advances in biomaterial technology allows the establishment of ex vivo expansion systems to overcome this bottleneck. The progress of various scaffold fabrication could direct stem cell fate decisions including cell proliferation and differentiation into specific lineages in vitro. Stem cell biology and biomaterial technology promote synergistic effect on stem cell-based regenerative therapies. Therefore, understanding the interaction of stem cell and biomaterials would allow the designation of new biomaterials for future clinical therapeutic applications for tissue regeneration. This review focuses mainly on the advances of natural and synthetic biomaterials in regulating stem cell fate decisions. We have also briefly discussed how biological and biophysical properties of biomaterials including wettability, chemical functionality, biodegradability and stiffness play their roles.     

Early events associated with periodontal connective tissue attachment formation on titanium and hydroxyapatite surfaces.

Endosseous dental implants can support at least three types of biomaterial/connective tissue interfaces: osseointegration, fibro-osseous integration, and periodontal connective tissue attachment. Although a periodontal connective tissue attachment offers distinct advantages, only osseointegration and fibro-osseous integration are at present clinically achievable. Recent studies indicate a periodontal regeneration-competent cell population and an appropriate biomaterial substrate both are required for periodontal connective attachment formation on biomaterial surfaces. We therefore have developed an in vitro model to characterize the effects of various biomaterial substrates on the early events of periodontal connective tissue attachment formation. Primary cultures of periodontal ligament and gingival connective tissue cells were cultured on uncoated (control) and coated (titanium- and hydroxyapatite-coated) tissue culture plastic, and the level of cell proliferation, collagen, and noncollagen protein synthesis, alkaline phosphatase activity, and expression of a 42 kD cementum extracellular matrix protein were measured over 5, 7, and 9 days in culture. While all three substrates supported cell attachment, proliferation, and protein synthesis, only uncoated and titanium-coated tissue culture plastic supported expression of the cementum extracellular matrix protein after 9 days of culture. In addition, the levels of cell proliferation and collagen and noncollagen protein synthesis for cells grown on hydroxyapatite-coated surfaces lagged behind cells cultured on the control or titanium-coated surfaces at each of the three time points. These data suggest that biomaterial substrates markedly can influence the temporal sequence of extracellular matrix proteins associated with periodontal connective tissue attachment formation. In addition to surface composition (titanium versus hydroxyapatite), surface properties (e.g., topography) also may have an effect on periodontal connective tissue attachment formation. This model may be of use in designing biomaterials to support the formation of periodontal connective tissue attachment in vivo.     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

Structural characteristics of small intestinal submucosa constructs dictate in vivo incorporation and angiogenic response

The rate of angiogenesis and cellular infiltration into degradable biomaterials determines scaffold persistence in vivo. The ability to tune the degradation properties of naturally derived biomaterials has been a popular goal in tissue engineering, yet has often depended on chemical crosslinking. Small intestinal submucosa (SIS) is a naturally derived, collagen-based, bioactive scaffold that has broad clinical success in many therapeutic applications. Two methods for producing multilayer, non-crosslinked SIS constructs were compared in vitro and in vivo. Traditional and cryo SEM, mercury intrusion porosimetry, and a novel enzymatic degradation assay determined that lyophilization produced an open, porous scaffold, in contrast to the collapsed, denser structure of SIS constructs produced using a vacuum press process. The angiogenic responses to lyophilized and vacuum-pressed SIS constructs were evaluated in vivo using a subcutaneous implant assay in mice. Explanted samples were compared after 7 and 21 days using fluorescence microangiography and light microscopy. Capacity of the implant neovasculature was also determined. These experiments revealed that the lyophilized SIS was infiltrated and vascularized more rapidly than the vacuum pressed. These data demonstrate the tunable incorporation of a non-crosslinked ECM-based biomaterial, which may have implications for the persistence of this degradable scaffold in tissue engineering.     

A perfusion bioreactor for engineering bone constructs: an in vitro and in vivo study

A perfusion bioreactor, which was designed based on fluidized bed concepts, was validated for the culture of bone constructs of clinically relevant size. For this study, natural coral has been used as three-dimensional scaffolds. This biomaterial is a microporous, biocompatible, osteoconductive, and absorbable scaffold. This perfusion bioreactor provided a stable environment in terms of osmolarity, pH, and, most importantly, oxidative stress. Bone constructs engineered in this system resulted in significantly higher cell proliferation and homogenous cell distribution than those cultured under static conditions. Particularly relevant to the production of bioengineered bone in a clinical setting, custom-made bone constructs (each one with volume up to 30 cm(3)) could be produced using a such perfusion bioreactor. Last, but not least, the bone constructs of clinically relevant volume thus produced were shown to be osteogenic when transplanted subcutaneously in sheep.     

A model for studying epithelial attachment and morphology at the interface between skin and percutaneous devices

Percutaneous devices are indispensable in modern medicine, yet complications from their use result in significant morbidity, mortality, and cost. Bacterial biofilm at the device exit site accounts for most infections in short-term devices. We hypothesize that advanced biomaterials can be developed that facilitate attachment of skin cells to percutaneous devices, forming a seal to preclude bacterial invasion. To study the skin/biomaterial interface systematically, we first identified biomaterials with physical properties compatible with histological processing of skin. Second, we developed an organ culture system to study skin response to implants. Organ cultures implanted with porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] or polytetrafluoroethylene (PTFE) could easily be evaluated histologically with preservation of the skin/biomaterial interface. Epithelial cells migrated down the cut edges of the biomaterial in a pattern seen in marsupialization of percutaneous devices in vivo. This in vitro model maintains skin viability and allows histologic evaluation of the skin/biomaterial interface, making this a useful, inexpensive test-bed for studies of epidermal attachment to modified biomaterials.     

Effects of topographical surface modifications of electron beam melted Ti-6Al-4V titanium on human fetal osteoblasts

The aim of the study was to assess the suitability of different Ti-6Al-4V surfaces produced by the electron beam melting (EBM) process as matrices for attachment, proliferation, and differentiation of human fetal osteoblasts (hFOB 1.19). Human osteoblasts were cultured in vitro on smooth and rough-textured Ti-6Al-4V alloy disks. By means of cell number and vitality and SEM micrographs cell attachment and proliferation were observed. The differentiation rate was examined by using quantitative real-time PCR analysis for the gene expression of alkaline phosphatase (ALP), type I collagen (Coll-I), bone sialoprotein (BSP) and osteocalcin (OC). After 3 days of incubation there was a significant higher vitality (p < 0.02) and proliferation (p < 0.02) of hFOB cells on smooth surfaces (R(a) = 0.077 microm) and compact surfaces with adherent partly molten titanium particles on the surface (R(a) /= 56.9 microm) reduced proliferation of hFOB cells. Surface characteristics of titanium can easily be changed by EBM in order to further improve proliferation.