In vitro and in vivo evaluation of biodegradable,open-porous scaffolds made of sintered magnesium W4 short fibres

A cytocompatible and biocompatible, degradable, open-porous, mechanically adaptable metal scaffold made of magnesium alloy W4 melt-extracted short fibres was fabricated by liquid phase sintering. Cylindrical samples (3 × 5 mm) of sintered W4 short fibres were evaluated under in vitro (L929, HOB, eudiometer, weight loss) and in vivo conditions (rabbits: 6 and 12 weeks). The in vitro corrosion environment (e.g., temperature, flow, composition of corrosion solution, exposure time) significantly influenced the corrosion rates of W4 scaffolds compared with corrosion in vivo. Corrosion rates under cell culture conditions for 72 h varied from 1.05 to 3.43 mm y^?1 depending on the media composition. Corrosion rates measured in eudiometric systems for 24 h were ～24–27 times higher (3.88–4.43 mm y^?1) than corrosion in vivo after 6 weeks (0.16 mm y^?1). Moreover, it was found that the cell culture media composition significantly influences the ionic composition of the extract by selectively dissolving ions from W4 samples or their corrosion products. A pilot in vivo study for 6 and 12 weeks demonstrated active bone remodelling, no foreign body reaction and no clinical observation of gas formation during W4 scaffold implantation. Long-term in vivo studies need to be conducted to prove complete degradation of the W4 scaffold and total replacement by the host tissue.     

The in vivo bone formation by mesenchymal stem cells in zein scaffolds

In our previous study, a three-dimensional zein porous scaffold was prepared. This scaffold showed proper mechanical properties, good biocompatibility, and controllable biodegradation. In addition, it allowed blood vessels to form inside in vivo. In the current study, we prepared the complexes of zein scaffolds and rabbit MSCs, and investigated ectopic bone formation in nude mice. Furthermore, we implanted them into the radius defects of rabbits and assessed whether they could be helpful in the repair of critical-sized bone defects. The results showed that the complexes of zein scaffolds and rabbit MSCs could undergo ectopic bone formation in the thigh muscle pouches of nude mice. More importantly, the complexes could lead to the repair of critical-sized radius defects in rabbits accompanied with blood vessels' formation, which clearly demonstrates promise for the treatment of bone defects through tissue engineering.     

In vitro and in vivo evaluation of porous PCL-PLLA 3D polymer scaffolds fabricated via salt leaching method for bone tissue engineering applications

Three dimensional porous scaffolds composed of various ratios of polycaprolactone and poly(L-lactic acid) (PLLA) were prepared using salt leaching method for bone regeneration applications. Surfaces of the scaffolds were visualized using scanning electron microscope (SEM) and the combination of the polymers was confirmed by FT-IR. Addition of PLLA increased the porosity and pore sizes of the scaffolds and also the scaffolds’ compressive strength initially. Osteoblast-like cells were used and it was found that the samples’ cell biocompatibility was further promoted with the increase in PLLA content as observed via cell proliferation assays using MTT, gene expression with RT-PCR, and micrographs from SEM and confocal microscopy. Samples were then implanted into male rabbits for 2 months, and histological staining and micro-CT histomorphometry show that new bone formations were detected in the site containing the implants of the scaffolds and that bone regeneration was further promoted with the increased concentration of PLLA in the scaffold.     

Characterization of mineralized collagen-glycosaminoglycan scaffolds for bone regeneration

Mineralized collagen–glycosaminoglycan scaffolds designed for bone regeneration have been synthesized via triple co-precipitation in the absence of a titrant phase. Here, we characterize the microstructural and mechanical properties of these newly developed scaffolds with 50 and 75 wt.% mineral content. The 50 wt.% scaffold had an equiaxed pore structure with isotropic mechanical properties and a Ca–P-rich mineral phase comprised of brushite; the 75 wt.% scaffold had a bilayer structure with a pore size varying in the through-thickness direction and a mineral phase comprised of 67% brushite and 33 wt.% monetite. The compressive stress–strain response of the scaffolds was characteristic of low-density open-cell foams with distinct linear elastic, collapse plateau and densification regimes. The elastic modulus and strength of individual struts within the scaffolds were measured using an atomic force microscopy cantilevered beam-bending technique and compared with the composite response under indentation and unconfined compression. Cellular solids models, using the measured strut properties, overestimated the overall mechanical properties for the scaffolds; the discrepancy arises from defects such as disconnected pore walls within the scaffold. As the scaffold stiffness and strength decreased with increasing overall mineral content and were less than that of natural, mineralized collagen scaffolds, these microstructural/mechanical relations will be used to further improve scaffold design for bone regeneration applications.     

Novel in vivo method for evaluation of healing around implants in bone

A material implanted in bone is always inserted into coagulating blood. Protein and cell interactions during this initial implantation time will govern later healing. Many studies have focused on the tissue surrounding implants. We have developed a method for evaluation of healing around implants in bone by studying cells adhering to the implant surface. Hydrophilic titanium discs were inserted into rat tibiae. Samples were retrieved after 1, 2, 4, and 8 days of implantation and were analyzed by fluorescence microscopy techniques and scanning electron microscopy. Both proliferating and apoptotic cells were found on the surface. Generally, cells closest to the implant surface were nonviable whereas cells in the fibrin network a distance from the surface were viable. Bone morphogenetic protein-2 (BMP-2) is an osteogenic substance. An increase in BMP-2-positive cells was seen during the implantation period, and a population of large BMP-2-positive cells appeared on the surface after 4 days of implantation. The method developed here is a suitable tool for rapid evaluation of the initial healing around implant material.     

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering

Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.     

灌注法制备全肾脏脱细胞基质支架的体内生物相容性

背景：应用灌注法制备的大鼠全肾脏脱细胞基质支架具有良好的体外细胞相容性,但其体内生物相容性尚不明确。 目的：应用灌注法制备大鼠全肾脏脱细胞基质支架,检测其体内生物相容性。 方法：应用灌注法制备Wistar大鼠全肾脏脱细胞基质支架,进行以下实验：①急性毒性实验：在小鼠腹腔分别注射全肾脏脱细胞基质支架浸提液、生理盐水及苯酚。②溶血实验：将抗凝新西兰兔血分别与全肾脏脱细胞基质支架浸提液、生理盐水及蒸馏水混合。③热源实验：向新西兰兔耳缘静脉注射全肾脏脱细胞基质支架浸提液。④内皮刺激实验：在新西兰兔皮下注射全肾脏脱细胞基质支架浸提液,观察有无皮肤刺激反应。⑤皮下植入实验：将全肾脏脱细胞基质支架埋入新西兰兔背部皮下。 结果与结论：全灌注法制备的Wistar大鼠全肾脏脱细胞基质支架无细胞残留,未引起全身毒性反应、急性溶血反应、热源反应及皮肤刺激反应,植入兔体内具有良好的组织相容性。说明大鼠全肾脏脱细胞基质材料在动物体内具有很好的生物相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Porous calcium polyphosphate scaffolds for bone substitute applications in vivo studies

Porous rods (6 mm in length and 4 mm in diameter) of calcium polyphosphate (CPP) made by gravity sintering of particles in the size ranges of 45-105, 105-150. and 150-250 microm and with initial volume percent porosity in the range of 35-45% were implanted in the distal femur of New Zealand white rabbits. In an initial experiment, four rabbits implanted with rods made from coarse particles (150-250 microm) were sacrificed at each of the following time points: 2 days, 2 weeks, 6 weeks and 12 weeks. In a subsequent experiment, 10 rabbits were implanted with rods made by sintering 45-105 microm particles and another 10 were made by using particles of 105-150 microm. These rabbits were sacrificed at 6 weeks (five rabbits) and 1 year (five rabbits). No adverse reaction was found histologically at any time point in either experiment. These experiments show that CPP macroporous rods can support bone ingrowth and that between 12 weeks and 1 year, the amount of bones formed is equivalent to the natural bone volume found at similar sites. The degradation of the CPP material is inversely proportional to the original particle size and is rapid initially (within the first 6 weeks) and slows down thereafter. In conclusion, this material seems to promote rapid bone ingrowth and can be tailored to degrade at a given rate in vivo to some degree through appropriate selection of the starting particle size.     

In vivo bone response to 3D periodic hydroxyapatite scaffolds assembled by direct ink writing

The in vivo bone response of 3D periodic hydroxyapatite (HA) scaffolds is investigated. Two groups of HA scaffolds (11 mm diameter x 3.5 mm thick) are fabricated by direct-write assembly of a concentrated HA ink. The scaffolds consist of cylindrical rods periodically arranged into four quadrants with varying separation distances between rods. In the first group, HA rods (250 microm in diameter) are patterned to create pore channels, whose areal dimensions are 250 x 250 microm(2) in quadrant 1, 250 x 500 microm(2) in quadrants 2 and 4, and 500 x 500 microm(2) in quadrant 3. In the second group, HA rods (400 microm in diameter) are patterned to create pore channels, whose areal dimensions of 500 x 500 microm(2) in quadrant 1, 500 x 750 microm(2) in quadrants 2 and 4, and 750 x 750 microm(2) in quadrant 3. Each group of scaffolds is partially densified by sintering at 1200 degrees C prior to being implanted bilaterally in trephine defects of skeletally mature New Zealand White rabbits. Their tissue response is evaluated at 8 and 16 weeks using micro-computed tomography, histology, and scanning electron microscopy. New trabecular bone is conducted rapidly and efficiently across substantial distances within these patterned 3D HA scaffolds. Our observations suggest that HA rods are first coated with a layer of new bone followed by subsequent scaffold infilling via outward and inward radial growth of the coated regions. Direct-write assembly of 3D periodic scaffolds composed of micro-porous HA rods arrayed to produce macro-pores that are size-matched to trabecular bone may represent an optimal strategy for bone repair and replacement structures.     

Quantitative detection of cell culture Mycoplasmas by a one step polymerase chain reaction method

A rapid, sensitive assay was developed that can detect the six species of Mycoplasmas that account for the vast majority of cell culture infections. This assay, a modification of the method published by Wong-Lee & Lovett [1], allows direct evaluation of culture medium by a single-step PCR method that utilizes primers complementary to conserved 16S rRNA sequences. Extensive testing of medium from uninfected cultures spiked with purified Mycoplasma DNAs showed that the method described in this report can detect the equivalent of one Mycoplasma in 15 l culture medium; thus, evaluation of a single culture sample allows detection of Mycoplasmas in cultures infected with the equivalent of 10 or more Mycoplasmas per 15 l (or 6.7×10² Mycoplasma equivalents/ml) with greater than 99.99% confidence. Comparison of results obtained with this PCR-based assay and a standard biological colony-forming assay revealed that the PCR assay is capable of detecting 0.0015-0.015 colony forming units, suggesting that the PCR assay may also be detecting nonviable Mycoplasmas. The high level of amplification achieved with this method allows direct detection of amplification products by ethidium bromide staining of agarose gels, and thus allows rapid screening of cell cultures.Abbreviations CFU colony forming unit - PCR polymerase chain reaction     

Kinetics of in vivo bone deposition by bone marrow stromal cells into porous calcium phosphate scaffolds: an X-ray computed microtomography study

In a typical bone tissue engineering application, osteogenic cells are harvested and seeded on a three-dimensional (3D) synthetic scaffold that acts as guide and stimulus for tissue growth, creating a tissue engineering construct or living biocomposite. Despite the large number of performed experiments in different laboratories, information on the kinetics of bone growth into the scaffolds is still scarce. Highly porous hydroxyapatite scaffolds were investigated before the implantation and after they were seeded with in vitro expanded bone marrow stromal cells (BMSC) and implanted for 8, 16, or 24 weeks in immunodeficient mice. Synchrotron x-ray computed microtomography (microCT) was used for qualitative and quantitative 3D characterization of the scaffold material and 3D evaluation of tissue engineered bone growth kinetics after in vivo implantation. Experiments were performed taking advantage of a dedicated set up at the European Synchrotron Radiation Facility (ESRF, Grenoble, France), which allowed quantitative imaging at a spatial resolution of about 5 microm. A peculiarity of these experiments was the fact that at first the data were obtained on the different pure scaffolds, then the same scaffolds were seeded by BMSC, implanted, and brought again to ESRF for investigating the formation of new bone. The volume fraction, average thickness, and distribution of the newly formed bone were evaluated as a function of the implantation time. New bone thickness increased from week 8 to week 16, but deposition of new bone was arrested from week 16 to week 24. Instead, mineralization of the newly deposited bone matrix continued up to week 24.     

In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method

Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from approximately 88 to approximately 405 microm) and porosity (from approximately 80% to approximately 94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380-405 microm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186-200 microm pore size was better for fibroblasts growth. Also the scaffold section with 290-310 microm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.     

In vitro and in vivo analysis of co-electrospun scaffolds made of medical grade poly(epsilon-caprolactone) and porcine collagen

In this study, a nanofiber mesh made by co-electrospinning medical grade poly(epsilon-caprolactone) and collagen (mPCL/Col) was fabricated and studied. Its mechanical properties and characteristics were analyzed and compared to mPCL meshes. mPCL/Col meshes showed a reduction in strength but an increase in ductility when compared to PCL meshes. In vitro assays revealed that mPCL/Col supported the attachment and proliferation of smooth muscle cells on both sides of the mesh. In vivo studies in the corpus cavernosa of rabbits revealed that the mPCL/Col scaffold used in conjunction with autologous smooth muscle cells resulted in better integration with host tissue when compared to cell free scaffolds. On a cellular level preseeded scaffolds showed a minimized foreign body reaction.     

Cytocompatibility of brushite and monetite cell culture scaffolds made by three-dimensional powder printing

This study investigated the cytocompatibility of low-temperature direct 3-D printed calcium phosphate scaffolds in vitro. The fabrication of the scaffolds was performed with a commercial 3-D powder printing system. Diluted phosphoric acid was printed into tricalcium phosphate powder, leading to the formation of dicalcium phosphate dihydrate (brushite). Hydrothermal conversion of the brushite matrices led to the formation of dicalcium phosphate anhydrous (monetite). The biocompatibility was investigated using the osteoblastic cell line MC3T3-E1. Cell viability and the expression of alkaline phosphatase served as parameters. The culture medium was analyzed for pH value, concentration of free calcium and phosphate ions and osteocalcin. Both types of scaffolds showed a considerable increase of cell proliferation and viability; the monetite matrices were a little inferior compared with the brushite ones. The activity of alkaline phosphatase showed a similar pattern. Optical and electron microscopy revealed an obvious cell growth on the surface of both materials. Analysis of the culture medium showed minor alterations of pH value within the physiological range. The concentrations of free calcium and phosphate ions were obviously different among brushite and monetite cultures, due to their different solubility. The content of osteocalcin of the culture medium was reduced by the printed scaffolds due to adsorption. We conclude that the powder printed brushite and monetite matrices have a suitable biocompatibility for their use as cell culture scaffolds. Both materials enable osteoblastic cells in vitro to proliferate and differentiate due to the expression of typical osteoblastic markers.     

Characterization and cytocompatibility of biphasic calcium phosphate/polyamide 6 scaffolds for bone regeneration

Porous scaffolds of biphasic calcium phosphate (BCP)/polyamide 6 (PA6) with weight ratios of 30/70, 45/55, and 55/45 have been fabricated through a modified thermally induced phase separation technique. The chemical structure properties, macrostructure, and mechanical strength of the scaffolds were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy, and mechanical testing. The results indicated that the BCP/PA6 scaffolds had an interconnected porous structure with a pore size mainly ranging from 100 to 900 μm and many micropores on the rough pore walls. The mechanical property of the scaffold was significantly enhanced by the addition of BCP inorganic fillers. The 55/45 BCP/PA6 composite scaffold with 76.5% ± 2.1% porosity attained a compressive strength of 1.86 ± 0.14 MPa. Moreover, the BCP/PA6 porous scaffold was cultured with rat calvarial osteoblasts to investigate the cell proliferation, viability, and differentiation function (alkaline phosphatase). The type I collagen expression was also used to characterize the differentiation of rat calvarial osteoblasts on BCP/PA6 composite scaffold by immunocytochemistry. The in vitro cytocompatibility evaluation demonstrated that the BCP/PA6 scaffold acted as a good template for the cells adhesion, spreading, growth, and differentiation. These results suggest that the BCP/PA6 porous composite could be a candidate as an excellent substitute for damaged or defect bone.     

In vivo response of polylactic acid-alginate scaffolds and bone marrow-derived cells for cartilage tissue engineering

Successful application of tissue-engineering techniques to damaged biological structures is determined by functional performance in vivo. This study evaluated the in vivo response of a tissue-engineered construct composed of a polylactic acid-alginate amalgam seeded with bone marrow-derived mesenchymal stem cells and stimulated in vitro with transforming growth factor beta for cartilage tissue engineering. Constructs were placed in cylindrical osteochondral defects in the canine femoral condyle and examined 6 weeks postoperatively by gross, histological, immunohistochemical, and biomechanical analyses. In the course of 6 weeks in vivo, the defects filled with a cartilaginous tissue regardless of whether cell-seeded (experimental) or cell-free (control) constructs were implanted; however, the quality of the tissue differed between the experimental and control defects. Cell-seeded experimental defects showed more cartilage-like matrix quality, cell distribution, and proteoglycan staining. Biomechanically, experimental and control specimens exhibited similar behavior; however, both tissues were still immature compared with normal cartilage. The evidence accumulated in this study showed a modest acceleration of the in vivo healing of cell-seeded constructs but also demonstrated a reparative response of cell-free constructs. This finding suggests that the constructs prepared from the PLA-alginate amalgam may serve as a means for host cell attachment.