Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

A rapid and efficient method for expansion of human mesenchymal stem cells

During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs) in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of different medium compositions on the expansion and osteogenic differentiation of bone marrow-derived hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a density of 100 cells/cm2 had a significantly higher growth rate than at 5000 cell/cm2, which was further improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implantation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation. Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture conditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding the cells at 100 cells/cm2 in an alpha-minimal essential medium-based medium containing dexamethasone.     

Oscillatory perfusion seeding and culturing of osteoblast-like cells on porous beta-tricalcium phosphate scaffolds

Perfusion culture systems have proven to be effective bioreactors for constructing tissue engineered bone in vitro, but existing circuit-based perfusion systems are complicated and costly for conditioned culture due to the large medium volume required. A compact perfusion system for artificial bone fabrication using oscillatory flow is described here. Mouse osteoblast-like MC 3T3-E1 cells were seeded at 1.5 x 10(6) cells/100 microL and cultured for 6 days in porous ceramic beta-tricalcium phosphate scaffolds (10 mm in diameter, 8 mm in height) by only 1.5 mL culture media per scaffold. The seeding efficiency, cell proliferation, distribution and viability, and promotion of early osteogenesis by both a static and an oscillatory perfusion method were evaluated. The oscillatory perfusion method generated higher seeding efficiency, alkaline phosphatase activity, and scaffold cellularity (by DNA content) after 6 days of culture. Stereomicroscopic observation of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining and Calcein-AM/propidium iodide double staining also demonstrated homogeneous seeding, proliferation, and viability of cells throughout the scaffolds in the oscillatory perfusion system. By contrast, the static culture yielded polarized seeding and proliferation favoring the outer and upper scaffold surfaces, with only dead cells in the center of the scaffolds. Thus, these results suggest that the oscillatory flow condition not only allow a better seeding efficiency and homogeneity, but also facilitates uniform culture and early osteogenic differentiation. The oscillatory perfusion system could be a simple and effective bioreactor for bone tissue engineering.     

Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.     

Mesenchymal stem cell-seeded collagen matrices for bone repair: effects of cyclic tensile strain, cell density, and media conditions on matrix contraction in vitro

Type I collagen is the most abundant extracellular matrix protein in bone and contains arginine- glycine-aspartic acid sequences that promote cell adhesion and proliferation. We have previously shown that human mesenchymal stem cells (hMSCs) seeded in three-dimensional (3D) collagen gels upregulate BMP-2 mRNA expression in response to tensile strain, indicative of osteogenesis. Therefore, collagen could be a promising scaffold material for functional bone tissue engineering using hMSCs. However, high contraction of the collagen gels by hMSCs poses a challenge to creating large, tissue-engineered bone constructs. The effects of cyclic tensile strain, medium (with and without dexamethasone), and hMSC seeding density on contraction of collagen matrices have not been investigated. hMSCs were seeded in 3D collagen gels and subjected to cyclic tensile strain of 10% or 12% for 4 h/day at a frequency of 1 Hz in osteogenic-differentiating or complete MSC growth media for up to 14 days. Viability of hMSCs was not affected by strain or media conditions. While initial seeding density affected matrix contraction alone, there was a high interdependence of strain and medium on matrix contraction. These findings suggest a correlation between hMSC proliferation and osteogenic differentiation on collagen matrix contraction that is affected by media, cell-seeding density, and cyclic tensile strain. It is vital to understand the effects of culture conditions on collagen matrix contraction by hMSCs in order to consider hMSC-seeded collagen constructs for functional bone tissue engineering in vitro.     

MR assessment of osteogenic differentiation in tissue-engineered constructs

Bone marrow stromal cells (MSC) are a promising source of osteoprogenitor cells for bone tissue engineering. However, the population of the osteoprogenitor cells and their differentiation potentials change with the gender, age, and health of the donor. Development of a noninvasive method to assess osteogenic progression is critical for successful bone tissue regeneration. High-resolution magnetic resonance imaging (MRI) (at 11.7 T, with spatial resolution of 62.5 x 62.5 microm in 500 microm slices) is used in the present study to monitor osteogenic differentiation of tissue-engineered constructs prepared by seeding human bone MSCs on gelatin sponge scaffolds. Quantitative measurements of the MR relaxation times (T1, T2) and the apparent diffusion coefficient (ADC) were performed for four successive weeks on control tissue constructs and constructs exposed to osteogenic differentiation medium. The T1 and T2 relaxation times and ADC were found to decrease as osteogenic progression proceeded in samples exposed to osteogenic differentiation medium. At week 4, the T1, T2, and ADC of TE constructs were 1.81 +/- 0.11 s, 19.5 +/- 11.02 ms, and 1.01 +/- 0.47 x 10(3) mm(2)/s, respectively, for osteogenic differentiated constructs, significantly different from control constructs 2.22 +/- 0.08 s, 50.39 +/- 5.57 ms, and 1.86 +/- 0.18 x 107(3) mm(2)/s (p < 0.05). The MR parameters were also highly correlated with the cell seeding densities and alkaline phosphatase (ALP) activities of the osteogenic constructs. In conclusion, periodic measurements of MR parameters (T1, T2, and ADC) provide a promising method for noninvasive monitoring of the status of tissue-engineered bone growth and differentiation.     

Self-assembled composite matrix in a hierarchical 3-D scaffold for bone tissue engineering

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.     

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze-thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5 x 10(5) cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3 x 10(6) cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (+/- 13.6) microg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.     

Overexpression of GDF5 through an adenovirus vector stimulates osteogenesis of human mesenchymal stem cells in vitro and in vivo

The use of stem cells combined with gene therapy could be an important way to facilitate bone regeneration. In this study, the aim was to investigate the potential of growth and differentiation factor-5 (GDF5) to genetically manipulate human mesenchymal stem cells (hMSCs) for bone regeneration. Recombinant adenovirus Ad-GDF5 and Ad-GFP were constructed and identified, and the titer of both were determined. Third-passage hMSCs were infected with adenovirus, and the expression of GDF5 was confirmed by detection of GFP-positive cells, GDF5 mRNA levels, Western blotting, and enzyme-linked immunosorbent assay (ELISA). hMSCs at passage 3 were divided into four groups: (1) an experimental group infected with Ad-GDF5, (2) a positive control group cultured with osteogenic differentiation medium, (3) a control group infected with Ad-GFP cultured with standard medium, and (4) a blank control group cultured with standard medium. Evaluation of cell morphology and proliferation, analysis of the expression of genes related to osteogenic differentiation, von Kossa staining, and immunofluorescent staining of collagen I were used to investigate the osteogenesis of cells among the groups. After culturing the cells for 2 days under each corresponding condition, the cells were detached and subcutaneously injected into the backs of nude mice to evaluate bone formation. Samples were collected for histological staining, protein Western blotting, and micro-computer tomography. When infected with Ad-GDF5, hMSCs could overexpress GDF5 for a prolonged period in vitro and reach a concentration of 160 ng/ml. Cells infected with Ad-GDF5 or cultured in osteogenic medium displayed osteogenic differentiation based on their histological and cellular properties and on their gene and protein expression patterns. Furthermore, Ad-GDF5 showed a better ability to upregulate the expression of collagen I, alkaline phosphatase, and osteocalcin mRNA than the osteogenic medium. Furthermore, Ad-GDF5 expression was associated with enhanced bone formation in vivo. Our findings suggest that hMSCs infected with Ad-GDF5 can differentiate in an osteogenic direction and may be a promising cell source for bone regeneration.     

人骨髓间充质干细胞的生物学特性及向神经前体细胞分化的实验研究

目的 观察体外培养的人骨髓间充质干细胞(hMSCs)的生物学特性,并探讨使其转分化为神经前体细胞(NPCs)的方法.方法以密度梯度离心和贴壁法相结合分离成人骨髓间充质干细胞,并观察细胞形态、生长、表面标记以及成骨和成软骨及成脂肪能力的情况.选用第3代细胞进行诱导,先经胚胎干细胞培养液扩增,再用加有5-氮胞苷和曲古菌素A的神经诱导液诱导,7d后,一部分样本进行Nestin、Sox2免疫荧光染色和RT-PCR检测;另一部分样本在含有B27的神经培养液中继续培养7d,然后进行NF-L的免疫荧光检测.结果分离培养的hMSCs纯度较高,CD29、CD44的阳性率均在90%以上;具有明显的成骨、成软骨和成脂肪能力;经5-氮杂胞苷和曲古菌素A作用后能向神经前体细胞分化,免疫荧光染色及RT-PCR结果显示,诱导后的细胞能特异性表达神经前体细胞标志物Nestin和Sox2;在神经培养液中继续培养后检测神经细胞标记物NF-L,可见较多阳性细胞.结论 hMSCs可在体外进行分离培养扩增,经药物修饰后具有向神经前体细胞分化的潜能.     

Continuing differentiation of human mesenchymal stem cells and induced chondrogenic and osteogenic lineages in electrospun PLGA nanofiber scaffold

Nanofibers have recently gained substantial interest for potential applications in tissue engineering. The objective of this study was to determine whether electrospun nanofibers accommodate the viability, growth, and differentiation of human mesenchymal stem cells (hMSCs) as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives. Poly(d,l-lactide-co-glycolide) (PLGA) beads with a PLA:PGA ratio of 85:15 were electrospun into non-woven fibers with an average diameter of 760+/-210 nm. The average Young's modulus of electrospun PLGA nanofibers was 42+/-26 kPa, per nanoindentation with atomic force microscopy (AFM). Human MSCs were seeded 1-4 weeks at a density of 2 x 10(6)cells/mL in PLGA nanofiber sheets. After 2 week culture on PLGA nanofiber scaffold, hMSCs remained as precursors upon immunoblotting with hKL12 antibody. SEM taken up to 7 days after cell seeding revealed that hMSCs, hMSC-Ob and hMSC-Ch apparently attached to PLGA nanofibers. The overwhelming majority of hMSCs was viable and proliferating in PLGA nanofiber scaffolds up to the tested 14 days, as assayed live/dead tests, DNA assay and BrdU. In a separate experiment, hMSCs seeded in PLGA nanofiber scaffolds were differentiated into chodrogenic and osteogenic cells. Histological assays revealed that hMSCs continuously differentiated into chondrogenic cells and osteogenic cells after 2 week incubation in PLGA nanofibers. Taken together, these data represent an original investigation of continuous differentiation of hMSCs into chondrogenic and osteogenic cells in PLGA nanofiber scaffold. Consistent with previous work, these findings also suggest that nanofibers may serve as accommodative milieu for not only hMSCs, but also as a 3D carrier vehicle for lineage specific cells.     

Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used.     

Perfusion cell seeding and cultivation induce the assembly of thick and functional hepatocellular tissue-like construct

Developing advanced technologies for encouraging the ex vivo assembly of functional hepatic tissue for implantation into the human body or for in vitro drug testing is one of the challenging tasks facing tissue engineers. In the present study, we utilized a perfusion bioreactor system equipped with a novel flow-distributing mesh for online cell seeding into macroporous alginate scaffolds and cultivation of multiple constructs of the C3A human hepatocyte cell line. Optimization of the medium flow rate (100 mL/min) and perfusion duration (12 h) yielded cell constructs with high cell seeding efficiency (98% of the input cells) and cell distribution throughout the entire scaffold. Further, we show that interstitial medium flow enabled uniform cell delivery into 35 constructs lined across the bioreactor cross section. Perfusion-cultivated cell constructs revealed much greater rates of cell proliferation, albumin-specific secretion, and gene expression of the phase I enzyme, CYP3A4, and phase II enzyme, UGT2B7, than did static-cultivated constructs. Most impressive was the 50-fold increase in CYP3A4 expression of the perfused cell constructs as compared to the level in static-cultivated cell constructs. We thus believe that the hepatic tissue constructs developed herein may be used in drug discovery programs for elucidating drug metabolism and toxicity profiles and for treating failing livers.     

Flow Perfusion Co-culture of Human Mesenchymal Stem Cells and Endothelial Cells on Biodegradable Polymer Scaffolds

In this study, we investigated the effect of flow perfusion culture on the mineralization of co-cultures of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). Osteogenically precultured hMSCs were seeded onto electrospun scaffolds in monoculture or a 1:1 ratio with HUVECs, cultured for 7 or 14 days in osteogenic medium under static or flow perfusion conditions, and the resulting constructs were analyzed for cellularity, alkaline phosphatase (ALP) activity and calcium content. In flow perfusion, constructs with monocultures of hMSCs demonstrated higher cellularity and calcium content, but lower ALP activity compared to corresponding static controls. ALP activity was enhanced in co-cultures under flow perfusion conditions, compared to hMSCs alone; however unlike the static controls, the calcium content of the co-cultures in flow perfusion was not different from the corresponding hMSC monocultures. The data suggest that co-cultures of hMSCs and HUVECs did not contribute to enhanced mineralization compared to hMSCs alone under the flow perfusion conditions investigated in this study. However, flow perfusion culture resulted in an enhanced spatial distribution of cells and matrix compared to static cultures, which were limited to a thin surface layer.     

Effect of seeding and bioreactor culture conditions on the development of human tissue-engineered cartilage

Human cartilage was produced using fetal chondrocytes seeded into polyglycolic acid (PGA) mesh scaffolds and cultured in recirculation bioreactors. The effect of scaffold thickness, seeding cell density, and bioreactor operating conditions on the quality of the engineered cartilage was investigated. Thin (2.15-mm-thick) PGA scaffolds lost their structural integrity during bioreactor culture and the resulting constructs were small and misshapen compared with tissues generated using 4.75-mm-thick scaffolds. Increasing the seeding cell number from 1.2 x 10(7) to 2.2 x 10(7 )per 4.75-mm-thick scaffold resulted in a doubling of the construct wet weight, a 4.4-fold increase in glycosaminoglycan (GAG) concentration, and a 2.9-fold increase in total collagen concentration in the tissues. Levels of GAG and total collagen were also improved significantly when 100 mL or 50% v/v of the culture medium was replaced periodically during operation of the bioreactors compared with 50, 25, or 5 mL. The proportion of GAG lost from the tissues into the medium was reduced by increasing the seeding cell number and replaced medium volume. This work demonstrates that the quality of tissue-engineered cartilage can be manipulated substantially depending on the cell seeding and bioreactor culture conditions employed.     

Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells

Adipose-derived human mesenchymal stem cells (hMSCs) will be more valuable for tissue engineering applications if they can be extensively subcultured without loss of phenotype and multilineage differentiation ability. This study examined the effects of serial passaging on growth rate, gene expression, and differentiation potential of adipose-derived hMSCs. Differentiation was assessed by analyzing changes in messenger RNA (mRNA) expression of osteogenic and adipogenic marker genes and by determining production of calcium deposits and lipid vacuoles. Cells cultured in osteogenic medium for 2 weeks upregulated expression of alkaline phosphatase mRNA relative to cells in growth medium, and deposited calcium. Calcium deposition decreased in cells from passages 4 to 6 but returned to levels near or above those of primary cells by passage 10. Cells cultured in adipogenic medium upregulated expression of lipoprotein lipase and peroxisome proliferator activated receptor-gamma mRNA relative to cells in growth medium, and formed lipid vacuoles at all passages. By passage 8, however, cells in adipogenic medium also deposited calcium. Growth rate was stable through passage 5, then decreased. The results of this study indicate that adipose-derived hMSCs are capable of both adipogenic and osteogenic differentiation through 10 passages (34 population doublings) but that osteogenic differentiation may start to dominate at later passages.     

Bioactive starch-based scaffolds and human adipose stem cells are a good combination for bone tissue engineering

Silicon is known to have an influence on calcium phosphate deposition and on the differentiation of bone precursor cells. This study explores the effect of the incorporation of silanol (Si-OH) groups into polymeric scaffolds on the osteogenic differentiation of human adipose stem cells (hASC) cultured under dynamic and static conditions. A blend of corn starch with polycaprolactone (30/70wt.%, SPCL) was used to produce three-dimensional fibre meshes scaffolds by the wet-spinning technique, and a calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups. In vitro assessment, using hASC, of functionalized and non-functionalized scaffolds was evaluated in either α-MEM or osteogenic medium under static and dynamic conditions (provided by a flow perfusion bioreactor). The functionalized materials, SPCL-Si, exhibit the capacity to sustain cell proliferation and induce their differentiation into the osteogenic lineage. The formation of mineralization nodules was observed in cells cultured on the SPCL-Si materials. Culturing under dynamic conditions using a flow perfusion bioreactor was shown to enhance the hASC proliferation and differentiation and a better distribution of cells within the material. The present work demonstrates the potential of these functionalized materials for future applications in bone tissue engineering. Additionally, these results highlight the simplicity, economic and reliable production process of those materials.     

Pro-osteogenic trophic effects by PKA activation in human mesenchymal stromal cells

Human mesenchymal stromal cells (hMSCs) are able to differentiate into a wide variety of cell types, which makes them an interesting source for tissue engineering applications. On the other hand, these cells also secrete a broad panel of growth factors and cytokines that can exert trophic effects on surrounding tissues. In bone tissue engineering applications, the general assumption is that direct differentiation of hMSCs into osteoblasts accounts for newly observed bone formation in vivo. However, the secretion of bone-specific growth factors, but also pro-angiogenic factors, could also contribute to this process. We recently demonstrated that secretion of bone specific growth factors can be enhanced by treatment of hMSCs with the small molecule db-cAMP (cAMP) and here we investigate the biological activity of these secreted factors. We demonstrate that conditioned medium contains a variety of secreted growth factors, with differences between medium from basic-treated and cAMP-treated hMSCs. We show that conditioned medium from cAMP-treated hMSCs increases proliferation of various cell types and also induces osteogenic differentiation, whereas it has differential effects on migration. Microarray analysis on hMSCs exposed to conditioned medium confirmed upregulation of pathways involved in proliferation as well as osteogenic differentiation. Our data suggests that trophic factors secreted by hMSCs can be tuned for specific applications and that a good balance between differentiation on the one hand and secretion of bone trophic factors on the other, could potentially enhance bone formation for bone tissue engineering applications.     

In vitro osteogenic differentiation of marrow stromal cells encapsulated in biodegradable hydrogels

Novel hydrogel materials based on oligo(poly(ethylene glycol) fumarate) (OPF) crosslinked with a redox radical initiation system were recently developed in our laboratory as injectable cell carriers for orthopedic tissue engineering applications. The effect of OPF hydrogel material properties on in vitro osteogenic differentiation of encapsulated rat marrow stromal cells (MSCs) with and without the presence of osteogenic supplements (dexamethasone) was investigated. Two OPF formulations that resulted in hydrogels with different swelling properties were used to encapsulate rat MSCs (seeding density approximately 13 million cells/mL, samples 6 mm diameter x 0.5 mm thick before swelling) and osteogenic differentiation in these constructs over 28 days in vitro was determined via histology and biochemical assays for alkaline phosphatase, osteopontin and calcium. Evidence of MSC differentiation was apparent over the culture period for samples without dexamethasone, but there was large variability in calcium production between constructs using cells of the same source. Differentiation was also seen in samples cultured with osteogenic supplements, but calcium deposition varied depending on the source pool of MSCs. By day 28, osteopontin and calcium results suggested that, in the presence of dexamethasone, OPF hydrogels with greater swelling promoted embedded MSC differentiation over those that swelled less (43.7 +/- 16.5 microg calcium/sample and 16.4 +/- 2.8 microg calcium/sample, respectively). In histological sections, mineralized areas were apparent in all sample types many microns away from the cells. These experiments indicate that OPF hydrogels are promising materials for use as injectable MSC carriers and that hydrogel swelling properties can influence osteogenic differentiation of encapsulated progenitor cells.