On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k_La value of the Tubespin at standard conditions was 24.3 h⁻¹, while that of a spinner flask was only 2.7 h⁻¹. The maximum cell density in the Tubespin bioreactor reached 6 × 10⁶ cells mL⁻¹, which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.     

The biocompatibility of calcium phosphate cements containing alendronate-loaded PLGA microparticles in?vitro

The composite of poly-lactic-co-glycolic acid (PLGA) and calcium phosphate cements (CPC) are currently widely used in bone tissue engineering. However, the properties and biocompatibility of the alendronate-loaded PLGA/CPC (APC) porous scaffolds have not been characterized. APC scaffolds were prepared by a solid/oil/water emulsion solvent evaporation method. The morphology, porosity, and mechanical strength of the scaffolds were characterized. Bone marrow mesenchymal stem cells (BMSCs) from rabbit were cultured, expanded and seeded on the scaffolds, and the cell morphology, adhesion, proliferation, cell cycle and osteogenic differentiation of BMSCs were determined. The results showed that the APC scaffolds had a porosity of 67.43 ± 4.2% and pore size of 213 ± 95 µm. The compressive strength for APC was 5.79 ± 1.21 MPa, which was close to human cancellous bone. The scanning electron microscopy, cell counting kit-8 assay, flow cytometry and ALP activity revealed that the APC scaffolds had osteogenic potential on the BMSCs in vitro and exhibited excellent biocompatibility with engineered bone tissue. APC scaffolds exhibited excellent biocompatibility and osteogenesis potential and can potentially be used for bone tissue engineering.     

Propagation ofSpodoptera frugiperda cells (Sf9) and production of recombinant proteins with the baculovirus expression system using improved spinner flasks with membrane aeration

Summary It has been shown that the growth of Spodoptera frugiperda cells is significantly reduced or ceased under oxygen limiting culture conditions. This paper describes the use of a new membrane-aerated spinner flask which was compared to conventional surface-aerated spinner flasks with regard to growth of the insect cell line Sf9 and recombinant protein production after infection with baculovirus. Using a commercially available serum-free culture medium Sf9 cells reached highest cell densities (3×10⁶ ml^–1) in the membrane-aerated spinner flask. Production of recombinant protein was also influenced by the oxygen supply. In the membrane-aerated spinner flask and in a surface-aerated spinner flask with reduced filling volume more than 20000 U ml^–1 of a recombinant interleukin-2 variant were accumulated whereas only 100 U ml^–1 were produced in a surface-aerated spinner flask with insufficient oxygen supply. Sufficient oxygenation appears to be essential for proliferation of Sf9 cells as well as recombinant protein production after infection with baculovirus. Membrane oxygenation allows sufficient oxygen supply at high cell density and an at least 2.5 fold higher filling volume per spinner unit.     

Defined Essential 8? Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm² of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x10⁶ cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.     

Osteogenic differentiation and osteochondral tissue engineering using human adipose‐derived stem cells

Osteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose‐derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion. Osteogenesis was induced and mineralized deposits were present in tissues produced using PGA culture in osteogenic medium. For development of osteochondral constructs, scaffolds seeded with stem cells were precultured in either chondrogenic or osteogenic medium, sutured together, and cultured in dual‐chamber stirred bioreactors containing chondrogenic and osteogenic media in separate compartments. After 2 weeks, total collagen synthesis was 2.1‐fold greater in the chondroinduced sections of the composite tissues compared with the osteoinduced sections; differentiation markers for cartilage and bone were produced in both sections of the constructs. The results from the dual‐chamber bioreactor highlight the challenges associated with achieving simultaneous chondrogenic and osteogenic differentiation in tissue engineering applications using a single stem‐cell source. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.     

Comparisons of microcarrier cell culture processes in one hundred mini-liter spinner flask and fifteen-liter bioreactor cultures

Microcarrier cell culture process can be used to culture anchorange-dependent cells in large bioreactor vessels. The process performance in large bioreactors is usually less prominent than that in spinner flask vessels and bench scale reactors. In this study we investigated the microcarrier cell culture processes in 100?ml spinner flask and 15-liter bioreactor cultures, including the kinetics for cell attachment, cell growth and the production of Japanese encephaltilis vaccine strain (Beijing-1) virus. Under a fixed concentration of microcarrier and cell density used in inoculations, the attachment kinetics of Vero cells on Cytodex 1 microcarrier in a 15-liter bioreactor vessel was 2 folds slower than with 100?ml spinner flask culture. Virus replication in 15-liter bioreactor culture also revealed an approximately one day lag-time compared to 100?ml spinner flask culture. Findings presented herein provide valuable information for designing and operating microcarrier cell culture processes in large bioreactor vessels.     

Fluid mechanics of a spinner-flask bioreactor

Spinner-flask bioreactors have been used for the production of articular cartilage in vitro. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. The present research focuses on the experimental and numerical characterization of the flow field within a spinner flask operating under conditions used to produce cartilage. Laboratory experiments carried out in a scaled-up model bioreactor employ particle-image velocimetry (PIV) to determine velocity and shear-rate fields in the vicinity of the construct closest to the stir bar, in addition to turbulence properties. Numerical computations calculated using FLUENT, a commercial software package, simulate the flow field in the same model bioreactor under similar operating conditions. In the computations, scaffolds were modeled as both solid and porous media with different permeabilities and flow rates through various faces of the construct nearest the stir bar were examined.     

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.     

Effect of pulse frequency on the osteogenic differentiation of mesenchymal stem cells in a pulsatile perfusion bioreactor

Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E(2) were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.     

造血细胞体外悬浮培养和生物反应器开发

为解决造血细胞的静态培养中由浓度梯度引起的培养不稳定、环境不均一、难放大等问题,首先采用转瓶对脐血单个核细胞进行了悬浮培养研究,结果表明,悬浮培养中总细胞、集落和CD34^＋细胞的扩增都高于静态的方瓶培养。在测试了所用材料生物相容性的基础上,开发了可以控制溶氧和pH的生物反应器,并将其应用到造血细胞的批培养中,结果表明反应器的培养环境均一,可实现较高密度的培养,而且总细胞、集落和CD34^＋细胞的扩增都优于静态培养。大规模的反应器培养有利于解决临床应用中细胞数量不足的问题。     

Bioreactor magnetic fields do not affect growth and antibody productivity of GAP-A3 hybridomas

Summary GAP-A3 hybridoma cells grown in T-flasks exposed to the rotating magnetic field of a spinner flask stirrer base show no effect of stirrer speed in the range 0–150 rpm on their growth rate or antibody productivity. Similarly mechanically and magnetically driven spinner flasks at 48 and 70 rpm show no difference in these rates that may be attributed to the magnetic field.     

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells (hDPSCs) on four commercially available scaffold biomaterials.METHODS: hDPSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. hDPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureOss (Allograft), Cerabone (Xenograft), PLLA (Synthetic), and OSTEON II Collagen (Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy (SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase (ALP) activity assay, respectively.RESULTS: All scaffold biomaterials except SureOss (Allograft) supported hDPSC adhesion, proliferation and differentiation. hDPSCs seeded on PLLA (Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hDPSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA (Synthetic) and OSTEON II Collagen (Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone (Xenograft) and OSTEON II Collagen (Composite) scaffolds, the hDPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape.CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hDPSCs. Hence, it may be useful in combination with hDPSCs for cell-based reconstructive therapy.     

Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature

Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35°C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20°C as those cultured at 25°C. Both desaturases had negligible activity in seeds cultured at 35°C compared to seeds cultured at 20°C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35°C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20°C cultures. Activities were twofold lower at 25°C and a further threefold lower in 35°C cultures. Cultures from 25 and 35°C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20°C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not.     

Abundance and Survival Rates of the Hawai’i Island Associated Spinner Dolphin (Stenella longirostris) Stock

Reliable population estimates are critical to implement effective management strategies. The Hawai’i Island spinner dolphin (Stenella longirostris) is a genetically distinct stock that displays a rigid daily behavioural pattern, foraging offshore at night and resting in sheltered bays during the day. Consequently, they are exposed to frequent human interactions and disturbance. We estimated population parameters of this spinner dolphin stock using a systematic sampling design and capture–recapture models. From September 2010 to August 2011, boat-based photo-identification surveys were undertaken monthly over 132 days (>1,150 hours of effort; >100,000 dorsal fin images) in the four main resting bays along the Kona Coast, Hawai’i Island. All images were graded according to photographic quality and distinctiveness. Over 32,000 images were included in the analyses, from which 607 distinctive individuals were catalogued and 214 were highly distinctive. Two independent estimates of the proportion of highly distinctive individuals in the population were not significantly different (p = 0.68). Individual heterogeneity and time variation in capture probabilities were strongly indicated for these data; therefore capture–recapture models allowing for these variations were used. The estimated annual apparent survival rate (product of true survival and permanent emigration) was 0.97 SE±0.05. Open and closed capture–recapture models for the highly distinctive individuals photographed at least once each month produced similar abundance estimates. An estimate of 221±4.3 SE highly distinctive spinner dolphins, resulted in a total abundance of 631±60.1 SE, (95% CI 524–761) spinner dolphins in the Hawai’i Island stock, which is lower than previous estimates. When this abundance estimate is considered alongside the rigid daily behavioural pattern, genetic distinctiveness, and the ease of human access to spinner dolphins in their preferred resting habitats, this Hawai’i Island stock is likely more vulnerable to negative impacts from human disturbance than previously believed.     

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP_i). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2P_i). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP_i-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.     

A Portable Anaerobic Microbioreactor Reveals Optimum Growth Conditions for the Methanogen Methanosaeta concilii

Conventional studies of the optimum growth conditions for methanogens (methane-producing, obligate anaerobic archaea) are typically conducted with serum bottles or bioreactors. The use of microfluidics to culture methanogens allows direct microscopic observations of the time-integrated response of growth. Here, we developed a microbioreactor (μBR) with ~1-μl microchannels to study some optimum growth conditions for the methanogen Methanosaeta concilii. The μBR is contained in an anaerobic chamber specifically designed to place it directly onto an inverted light microscope stage while maintaining a N₂-CO₂ environment. The methanogen was cultured for months inside microchannels of different widths. Channel width was manipulated to create various fluid velocities, allowing the direct study of the behavior and responses of M. concilii to various shear stresses and revealing an optimum shear level of ~20 to 35 μPa. Gradients in a single microchannel were then used to find an optimum pH level of 7.6 and an optimum total NH₄-N concentration of less than 1,100 mg/liter (<47 mg/liter as free NH₃-N) for M. concilii under conditions of the previously determined ideal shear stress and pH and at a temperature of 35°C.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Trehalose Maintains Bioactivity and Promotes Sustained Release of BMP-2 from Lyophilized CDHA Scaffolds for Enhanced Osteogenesis In Vitro and In Vivo

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ±5.32%) and area (40.71% ±7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ±0.44%, calcein: 6.08% ±1.37%) and mineral apposition rate (4.13±0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17±15.02 Mpa) and load of fracture (144.67±16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.