TLR7 Agonist-Loaded Gadolinium Oxide Nanotubes Promote Anti-Tumor Immunity by Activation of Innate and Adaptive Immune Responses.

Improving the delivery of biomolecules to DCs and lymph nodes is critical to increasing their anti-tumor efficacy, reducing their off-target side effects, and improving their safety. In this study, Gd2O3 nanotubes with lengths of 70-80 nm, diameters of 20-30 nm, and pore sizes of up to 18 nm were synthesized using a facile one-pot solvothermal method. The Gd2O3 nanotubes showed good adsorption capacity of OVA and TLR7a, with a loading efficiency of about 100%. The Gd2O3 nanotubes showed pH-sensitive degradation and biomolecule release properties; the release of gadolinium ions, OVA, and TLR7a was slow at pH 7.4 and fast at pH 5. The Gd2O3 nanotubes showed 2.6-6.0 times higher payload retention around the injection site, 3.1 times higher cellular uptake, 1.7 times higher IL1ß secretion, 1.4 times higher TNFα secretion by BMDCs, and markedly enhanced draining lymph node delivery properties. The combination of OVA, TLR7a, and Gd2O3 nanotubes significantly inhibited tumor growth and increased survival rate compared with only OVA-TLR7a, only OVA, and saline. The Gd2O3 nanotubes are biocompatible and can also be used as radiation sensitizers.

Induction of angiogenesis and neural progenitor cells by basic fibroblast growth factor-releasing polyglycolic acid sheet following focal cerebral infarction in mice.

Biodegradable sheets loaded with basic fibroblast growth factor (bFGF) are prepared as novel bFGF-releasing systems from polyglycolic acid nonwoven fabric by oxygen plasma treatment followed by bFGF adsorption. In the present study, we investigated the therapeutic effects of this system on a focal cerebral infarction model (CB-17 mouse). A preliminary in vitro study showed that this system released bFGF in an acellular culture medium, thereby keeping the bFGF concentration in the medium at ≥5 ng/ml for a prolonged period of 7 days. The released bFGF from this system retained its biological activity to enhance endothelial tube formation in vitro. In a mouse model of subacute focal cerebral infarction, this system increased the expression of endogenous vascular endothelial growth factor in the peri-infarct cortex and subventricular zone, promoted angiogenesis in the striatum, and increased neural progenitor cells in the peri-infarct cortex. Thus, this bFGF-releasing system has the potential to be a novel therapeutic approach for cerebral infarction.

The enhancing effects of heparin on the biological activity of FGF-2 in heparin-FGF-2-calcium phosphate composite layers.

Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the composite layers is important for its wider application in orthopedics and dentistry. This study incorporated low-molecular-weight heparin (LMWH) into the FGF-2-calcium phosphate composite layers and clarified the enhancing effects of LMWH on the biological activity of FGF-2 in the composite layers in vitro. LMWH-FGF-2-calcium phosphate composite layers were successfully formed on zirconia in supersaturated calcium phosphate solutions. The composite layers comprised continuous and macroscopically homogeneous layers and particles smaller than 500 nm in size composed of amorphous calcium phosphate. The amounts of Ca and P deposited on zirconia remained almost unchanged with the addition of LMWH under the presence of FGF-2 in the supersaturated calcium phosphate solution. The LMWH in the supersaturated calcium phosphate solution increased the stability of FGF-2 in the solution and the amount of FGF-2 in the composite layers. The LMWH in the composite layers increased the mitogenic and endothelial tube-forming activities of FGF-2, and FGF-2 activity of inducing osteogenic differentiation gene expression pattern in the composite layers. Our results indicate that the enhanced biological activity of FGF-2 in the LMWH-FGF-2-calcium phosphate composite layers is attributed to an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the supersaturated calcium phosphate solution and the composite layers. The LMWH-FGF-2-calcium phosphate composite layer is a promising coating for orthopedic and dental implants. STATEMENT OF SIGNIFICANCE: Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the layers is important for wider its application in orthopedics and dentistry. This study demonstrates the enhancing effects of low-molecular-weight heparin (LMWH) contained within LMWH-FGF-2-calcium phosphate composite layers on the biological activity of FGF-2 in vitro. Our results indicate that the enhanced biological activity of FGF-2 within the composite layers arises from an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the LMWH-FGF-2-calcium phosphate composite layers and supersaturated calcium phosphate solutions used for coating the composite layers.

Cell attachment area of rat mesenchymal stem cells correlates with their osteogenic differentiation level on substrates without osteoconductive property.

Cell morphology is related to proliferation and differentiation. We previously reported that cell attachment area of rat mesenchymal stem cells (MSCs) is negatively correlated with their osteogenic differentiation level on osteoconductive hydroxyapatite (HAp) with various microstructures. In this study, the correlation between the cell attachment area and osteogenic differentiation level was investigated on substrates without osteoconductive property using tissue culture polystyrene (TCPS), and 3 mol% yttria-stabilized zirconia (3Y-TZP) with or without surface periodic microstructures. It was found that the osteogenic differentiation level after 3 weeks of culture increased with a decrease in cell attachment area after 3 h of culture. The square of the correlation coefficient between cell attachment area and osteocalcin secretion content was 0.845 among the three types of substrates. Thus, the negative correlation between cell attachment area and differentiation level is confirmed even when cultured on substrates without osteoconductive property. These findings suggest that the correlation between the cell attachment area of rat MSCs and osteogenic differentiation level could also apply to various types of substrate, regardless of osteoconductive property.

Initial clinical trial of pins coated with fibroblast growth factor-2-apatite composite layer in external fixation of distal radius fractures.

BACKGROUND: Pin tract infection and loosening are major complications and challenges in the treatment of fractures by external fixation. To address this issue, we developed titanium pins coated with a fibroblast growth factor 2 (FGF-2)-apatite composite layer. The purpose of this initial clinical trial is to clarify the safety and feasibility of using these pins for the external fixation of distal radius fractures. METHODS: Unstable, displaced fractures of the distal radius that were medically suitable for external fixation were treated using external fixation pins coated and uncoated with an FGF-2-apatite composite layer. The coated pin group (n = 5) comprised 5 women (average age, 70.4 ±â€¯5.9 years), whereas the uncoated pin group (n = 10) comprised 8 women and 2 men (average age, 64.4 ±â€¯11.7 years). The average duration of external fixation was 40.8 ±â€¯1.3 and 41.6 ±â€¯2.1 days for the coated and uncoated pin groups, respectively. RESULTS: All patients achieved fracture union. One patient in the uncoated group had severe pin tract infection on the day of pin extraction. No pin loosening or difficulty in pin removal was observed in either group. Bacterial growth was present in 5% and 25% of the pin sites in the coated and uncoated groups, respectively (p = 0.059). No adverse events such as tumor formation were observed for more than 2 years after surgery in the coated pin group. CONCLUSIONS: This study clarified the safety and feasibility of using pins coated with an FGF-2-apatite composite layer for the external fixation of distal radius fractures.

Rod-shaped and substituted hydroxyapatite nanoparticles stimulating type 1 and 2 cytokine secretion.

A Th1 immune response is required for modern vaccines as the most commonly used alum adjuvant has weak capacity for inducing Th1 immune response. Herein, rod-shaped hydroxyapatite (HA), magnesium-substituted HA (MgHA) and zinc-substituted HA (ZnHA) nanoparticles with irregular nanopores were synthesized and used as immunoadjuvants. Magnesium and zinc substitution in HA showed no influence on morphology, particle size, zeta potential and surface area of the nanoparticles. The rod-shaped MgHA and ZnHA nanoparticles promoted the cellular uptake of a molecular immunopotentiator, stimulated both type 1 and 2 cytokine secretion in vitro that relate to Th1 and Th2 immunity of bone marrow dentritic cells, respectively. The MgHA and ZnHA nanoparticles may be useful as immunoadjuvants for human.

Gamma-cross-linked nonfibrillar collagen gel as a scaffold for osteogenic differentiation of mesenchymal stem cells.

We fabricated a transparent nonfibrillar collagen gel using gamma irradiation (5 kGy) and cultured rat mesenchymal stem cells (MSCs) on both the gamma-irradiated collagen gel and on unirradiated fibrillar collagen gel. Cells attached well and proliferated with high viability on the surface of both gels. The cells cultured on the gamma-irradiated nonfibrillar gel had a unique elongated shape and adhered to each other in culture. After 21 days of culture in dexamethasone-containing culture medium, the contents of bone-specific osteocalcin and calcium on the gamma-irradiated nonfibrillar gel were 1.4 and 1.9 times higher than those on fibrillar collagen gel, respectively. These data show that osteogenic differentiation of MSCs was promoted more efficiently on the gamma-cross-linked nonfibrillar gel than on the fibrillar gel and demonstrate the potential of the gamma-irradiated collagen gel for use in bone tissue engineering.

Correlation between cell attachment areas after 2 h of culture and osteogenic differentiation activity of rat mesenchymal stem cells on hydroxyapatite substrates with various surface properties.

The initial attachment of mesenchymal stem cells (MSCs) to substrates and osteogenic differentiation are supported by culture on a hydroxyapatite substrate. Cell attachment areas of rat MSCs after 2 h of culture on hydroxyapatite substrates with various microstructures and the osteogenic differentiation activity thereafter were measured. The perceived outcome was that, after 2 h of culture, rat MSCs with a small attachment area would have a high osteogenic differentiation activity, whereas those with a large attachment area would have a low osteogenic differentiation activity. Furthermore, rat MSCs with a small attachment area had many cytoplasmic processes, while those with a large attachment area revealed clear stress fibers and focal contacts. These results suggest that cell attachment area of rat MSCs after 2 h of culture has a strong effect on the osteogenic differentiation of rat MSCs. Thus, the measurement of cell attachment area after 2 h of culture could become valuable for estimating the osteogenic differentiation activity of rat MSCs thereafter.

DNA-lipid-apatite composite layers enhance gene expression of mesenchymal stem cells.

DNA-apatite composite layer (D-Ap layer) and DNA-lipid-apatite composite layer (DLp-Ap layer) were prepared on ceramic hydroxyapatite disk and scaffold using supersaturated calcium phosphate solutions supplemented with 0.5-5 µg/mL plasmid and 0-10 µL/mL lipid transfection reagent FuGENE®. Both in vitro and in vivo studies were carried out using mesenchymal stem cells (MSCs) and two kinds of gene (luciferase and bone morphogenetic protein (BMP)-2) for demonstrating potential application of the gene transfer system using the D-Ap and DLp-Ap layers in bone tissue engineering. In the in vitro study using luciferase gene, the DLp-Ap layers showed 1-2 orders of magnitudes higher gene transfer efficiency to MSCs than the D-Ap layer. In the in vivo study using BMP-2 gene, DLp-Ap layer slightly increased BMP-2 protein concentration than D-Ap layer, thereby enhancing their osteogenic differentiation than D-Ap layer. The present gene transfer system using the DLp-Ap layers, with the advantages of good biocompatibility, bone-bonding ability, and efficacy in in vitro and in vivo gene transfer to MSCs, would be useful in bone tissue engineering.

Bone morphogenetic protein-2 in biodegradable gelatin and ß-tricalcium phosphate sponges enhances the in vivo bone-forming capability of bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (MSCs) have been used for bone tissue engineering due to their osteogenic differentiation capability, but their application is controversial. To enhance their capability, we prepared biodegradable gelatin sponges incorporating ß-tricalcium phosphate ceramics (GT sponge), which has been shown to possess excellent controlled drug-release properties. The GT sponge was used as a carrier for both rat MSCs and bone morphogenetic protein-2 (BMP-2) and osteogenic differentiation was assessed by subcutaneous implantation of four different kinds of implants, i.e. GT-alone, MSC-GT composites, BMP-GT composites and BMP-GT composites supplemented with MSCs (BMP-MSC-GT) in rats. Two weeks after implantation, histological sections showed new bone formation in the peripheral parts of the BMP-GT and in almost the total volume of the BMP-MSC-GT implants. After 4 weeks, histology as well as microCT analyses demonstrated extensive bone formation in BMP-MSC-GT implants. Gene expression and biochemical analyses of both alkaline phosphatase and bone-specific osteocalcin confirmed the histological findings. These results indicate that the combination of MSCs, GT and BMP synergistically enhances osteogenic capability and provides a rational basis for their clinical application in bone reconstruction.

Development of an early estimation method for predicting later osteogenic differentiation activity of rat mesenchymal stromal cells from their attachment areas.

Cell morphology has received considerable attention in recent years owing to its possible relationship with cell functions, including proliferation, differentiation, and migration. Recent evidence suggests that extracellular environments can also mediate cell functions, particularly cell adhesion. The aims of this study were to investigate the correlation between osteogenic differentiation activity and the morphology of rat mesenchymal stromal cells (MSCs), and to develop a method of estimating osteogenic differentiation capability of MSCs on biomaterials. We measured the attachment areas of MSCs on substrates with various types of surface after 2 h of seeding, and quantified the amount of osteocalcin secreted from MSCs after 3 weeks of culture under osteogenic differentiation conditions. MSCs with small attachment areas showed a high osteogenic differentiation activity. These findings indicate that cell attachment areas correlate well with the osteogenic differentiation activity of MSCs. They also suggest that the measurement of cell attachment areas is useful for estimating the osteogenic differentiation activity of MSCs and is a practical tool for applications of MSCs in regenerative medicine.

In vivo study of dendronlike nanoparticles for stem cells "tune-up": from nano to tissues.

The control of stem cell differentiation to obtain osteoblasts in vivo is still regarded as a challenge in stem-cell-based and bone-tissue engineering strategies. Biodegradable dexamethasone-loaded dendron-like nanoparticles (NPs) of carboxymethylchitosan/poly(amidoamine) dendrimer have been proposed as intracellular drug-delivery systems of bioactive molecules. In this study, combination of nanotechnology, stem-cell engineering and tissue engineering is proposed in pre-programming the fate of rat bone marrow stromal cells (RBMSCs) towards osteoblasts cells and development of new bone tissue, in vivo. This work demonstrated that the developed NPs were able to be taken up by RBMSCs, and exhibited a noncytotoxic behavior in vitro. The performance of the developed dendronlike NP system for the intracellular delivery of dexamethasone was investigated by seeding the engineered RBMSCs onto starch-polycaprolactone scaffolds ex vivo, and implanting subcutaneously in the back of Fischer 344/N rats (Syngeneic), in the absence of the typical osteogenic supplements. Favorable results were observed in vivo, thus suggesting that stem cell "tune-up" strategy can open up a new regenerative strategy for bone-tissue engineering. FROM THE CLINICAL EDITOR: In this study, a combination of nanotechnology, stem-cell engineering and tissue engineering is proposed in pre-programming the fate of rat bone marrow stromal cells (RBMSCs) towards osteoblasts cells and development of new bone tissue in vivo.

In vitro osteogenic differentiation of HOS cells on two types of collagen gels.

HOS cell is a model strain of human osteoblasts derived from human osteosarcoma. We cultured the HOS cells on both the conventional collagen gel (neutral gel), and the gamma-crosslinked collagen gel without collagen fibrils (acidic gel). The shape of HOS cells on the neutral gel was similar to that on the culture dish. However, HOS cells on acidic gel had an elongated shape and attached each other to form a mesh-like pattern. The cells attached to the surface of both gels but scarcely penetrated their depths. We measured the biochemical markers for osteogenic differentiation in the HOS cells cultured on both the neutral gel and the acidic gel. The expressions of alkaline phosphatase and osteocalcin were detected in the HOS cells on both types of collagen gel. Deposition of the calcium also occurred on both gels although it was higher in the neutral gel than the acidic one. These results indicate the importance of collagen for the differentiation of HOS cells, but it is not dependent on the molecular structure (fibril formation) of collagen.

Hard tissue-forming potential of stem/progenitor cells in human dental follicle and dental papilla.

OBJECTIVE: The existence of stem/progenitor cells in dental tissue has been suggested but their characterization in the human tooth germ remains elusive. The purpose of this study was to investigate these cells in human dental follicles and dental papillae at the crown-forming stage and compare their potential for hard tissue formation. DESIGN: We used dental follicle cells (DFCs) and dental papilla cells (DPCs) derived from dental follicles and dental papillae at the crown-forming stage and compared their proliferative capacity, cell surface antigens and ability to form hard tissue in vitro and in vivo. RESULTS: Both DFCs and DPCs had extensive proliferation ability, expressed similar cell surface antigens and were capable of forming hard tissue in vivo as well as in vitro. However, there were two differences between DFCs and DPCs. First, DPCs had a significantly higher calcium accumulation than that in DFCs. Second, DFCs expressed a cementoblast marker, whereas DPCs expressed an odontoblast marker. CONCLUSIONS: We propose that dental follicles and dental papillae at the crown-forming stage contain different types of stem/progenitor cells and may have hard tissue-forming ability in a possibly origin-specific lineage direction.

Bone augmentation by bone marrow mesenchymal stem cells cultured in three-dimensional biodegradable polymer scaffolds.

Poly-lactic-glycolic acid (PLGA) is a biocompatible as well as biodegradable polymer and used in various medical applications. In this study, we evaluated efficiency of the specially designed three-dimensional porous PLGA as a scaffold for bone augmentation. First, cell attachment/proliferation, differentiation, and mineralization of Fisher 344 rat marrow mesenchymal stem cells (MSCs) cultured on the PLGA scaffold were analyzed. Viable MSCs were impregnated into pore areas of the scaffold and a moderate increase of DNA contents was seen. High alkaline phosphatase, osteocalcin content, and calcium content of MSCs in PLGA scaffolds under osteogenic differentiation conditions were seen after 14 or 21 days of culture. Subsequently, we implanted the PLGA/MSCs composites on rat calvaria bone for 30 days. Newly formed bone was seen in only the composite PLGA/MSCs implantation group, which had been precultured under osteogenic condition. We also demonstrated that the newly formed bone originated from the donor composites. These results demonstrate that the three-dimensional PLGA scaffold can support osteogenic differentiation of MSCs, and the scaffold combined with osteogenic MSCs can be used for in vivo bone tissue augmentation.

The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles.

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.

Macroporous hydroxyapatite scaffolds for bone tissue engineering applications: physicochemical characterization and assessment of rat bone marrow stromal cell viability.

In this work, a new methodology is reported for developing hydroxyapatite (HA) scaffolds using an organic sacrifice template. The novelty of work consists of possibility of obtaining porous and highly interconnected scaffolds mimicking the sacrificial component. Our purpose consisted of evaluating the physicochemical properties of the HA scaffolds by means of Fourier transform infra-red spectroscopy, X-ray diffraction analysis, and scanning electron microscopy (SEM) attached with an X-ray detector. The HA scaffolds obtained possess a porosity of approximately 70%, and macropores diameter in the range of 50-600 microm. In contrast, results regarding the microcomputed tomography analysis have demonstrated both high pore uniformity and interconnectivity across the scaffolds. The compressive strength of the HA scaffolds was found to be 30.2 +/- 6.0 MPa. Bioactivity of the HA scaffolds was assessed by immersion into a simulated body fluid solution, in vitro. SEM observations have showed a deposition of apatite on the surface of the HA scaffolds, with a "cauliflower-like" morphology after 1 day, and tend to be more pronounced with the immersion time. The changes in calcium and phosphorus concentration were monitored by inductively-coupled plasma optical emission spectrometry. Cytotoxicity of the HA scaffolds was preliminarily investigated by carrying direct observation of mouse fibroblasts cells (L929 cell-line) death in the inverted microscope, and then cell viability was determined by means of carrying out a MTS assay. Complementarily, a luminescent cell viability assay based on the quantification of adenosine triphosphate was performed using rat bone marrow stromal cells (RBMSCs). A LIVE/DEAD assay and SEM analysis allowed the visualization of the RBMSCs adhesion and proliferation on the surface of the HA scaffolds. According to the results obtained from 3D architecture, mechanical properties, biocompatibility, and adhesion tests, it is suggested that HA scaffolds has potential to find applications in bone tissue engineering scaffolding.

In vivo survival and osteogenic differentiation of allogeneic rat bone marrow mesenchymal stem cells (MSCs).

Marrow mesenchymal stem cells (MSCs) are multipotent progenitor cells and reported to be immunoprivileged as well as immunosuppressive. Hence, MSCs might be ideal candidates for allogeneic transplantation to induce regeneration of damaged tissues/organs. To confirm the differentiation capability of allogeneic MSCs in vivo is important for the acceleration of regenerative medicine. Consequently, we have established an in vivo rat model using subcutaneous implantation of a hydroxyapatite (HA) ceramic/MSCs composite. Osteogenic differentiation was used as an indicator of differentiation. When syngeneic MSCs were implanted, MSCs showed osteogenic differentiation as evidenced by new bone formation as well as high alkaline phosphatase (ALP) activity. When allogeneic MSCs were implanted, none of the allografts survived or showed osteogenic differentiation. However, when the recipient rats were treated with FK506 immunosuppressant, allogeneic MSCs showed osteogenic differentiation. Although this finding might not be adequate for the acceleration of regenerative medicine, these results did confirm that MSCs are not intrinsically immunoprivileged but that under appropriate immunosuppressant treatment, allogeneic MSCs can survive and show differentiation capability in vivo.

Small interfering RNA of alkaline phosphatase inhibits matrix mineralization.

To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.

Comparison of osteogenic ability of rat mesenchymal stem cells from bone marrow, periosteum, and adipose tissue.

Mesenchymal stem cells (MSCs) reside in many types of tissue and are able to differentiate into various functional cells including osteoblasts. Recently, adipose tissue-derived MSCs (AMSCs) have been shown to differentiate into many lineages, and they are considered a source for tissue regeneration. The purpose of this study was to compare the osteogenic differentiation capability of MSCs from bone marrow (BMSCs), MSCs from periosteum (PMSCs), and AMSCs using in vitro culture and in vivo implantation experiments. We harvested these MSCs from 7-week-old rats. The cells were seeded and cultured for 7 days in primary culture to assay a colony-forming unit. The frequency of the unit was the smallest in the BMSCs (P < 0.001). After primary culture, subculture was performed under osteogenic differentiation conditions for 1 and 2 weeks to detect mineralization as well as the bone-specific proteins of alkaline phosphatase and osteocalcin as osteogenic markers. BMSCs and PMSCs showed distinct osteogenic differentiation capability in comparison with other MSCs (P < 0.001). For the in vivo assay, composites of these cells and hydroxyapatite ceramics were subcutaneously implanted into syngeneic rats and harvested after 6 weeks. Micro-computed tomographic (CT) and histological analyses demonstrated that new bone formation was detected in the composites using BMSCs and PMSCs, although it was hard to detect in other composites. The CT analyses also demonstrated that the bone volume of BMSC composites was more than that of AMSC composites (P < 0.001). These results indicate that BMSCs and PMSCs could be ideal candidates for utilization in practical bone tissue regeneration.