Proliferation and harvest of human mesenchymal stem cells using new thermoresponsive nanocomposite gels.

For tissue engineering and regenerative medicine, stem cells should be effectively cultured in vitro. New thermoresponsive nanocomposite gels (MD-NC gels), consisting of inorganic clay (hectorite) and copolymers composed of hydrophobic 2-methoxyethyl acrylate (MEA) and hydrophilic N,N-dimethylacrylamide (DMAA) units, could be applied in cell culture and cell harvesting without trypsinization, specifically using mesenchymal stem cells (MSCs). The composition of the MD-NC gel (the ratio of the two monomer types and the clay content) was found to determine its swelling properties in the culture medium, thermosensitivity, protein adsorption, and cell attachment and proliferation. Various kinds of human cells, including MSCs, osteoblast (HOS) cells, fibroblast (NHDF) cells, and epithelial cells could be effectively cultured on MD-NC gels. In particular, on an MD10-NC2 gel with relatively low DMAA and clay content, the cells could be harvested by decreasing the temperature, either as a cell sheet (MSCs or NHDF cells) or as a population of suspension cells (HOS cells). Further, it was found that the MD10-NC2 gel is suitable for stem cell differentiation. Because of their thermosensitivity, controllable modulus, and surface properties, MD-NC gels are promising cell culture substrates useful for tissue engineering and regenerative medicine.

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.

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.

Thermoresponsible cell adhesion/detachment on transparent nanocomposite films consisting of poly(2-methoxyethyl acrylate) and clay.

Soft, transparent and mechanically tough nanocomposite (M-NC) films composed of poly(2-methoxyethyl acrylate) (PMEA) and inorganic clay (hectorite) were studied as substrates for a living cell harvest system. It was found that five cell types could all be cultivated to confluence on the surface of M-NC n films with clay content (n = 10-23 wt%), although the cells hardly cultivated on the surface of chemically-cross-linked PMEA and linear PMEA films. Further, it was found that the cells cultured on the surfaces of M-NC films can be detached, without any enzymatic digestion, by decreasing the medium temperature and/or simultaneously using gentle pipetting. The detached cell was obtained as a single cell or a contiguous cell sheet, both of which were viable and recultured. From the compositions and surface properties, it was estimated that the cell culture and subsequent cell detachment were attributed to the unique PMEA/clay network. The new soft nanocomposite is potentially a very promising substrate for tissue engineering.

Effects of epigallocatechin gallate on osteogenic capability of human mesenchymal stem cells after suspension in phosphate-buffered saline.

Administration of culture-expanded mesenchymal stem cells (MSCs) has been sought for regeneration of various damaged tissues/organs in clinical situations. Liquid suspensions of MSCs have either been directly injected into organs or generally infused. In this study, we focused on the viability and differentiation of MSCs after suspension in phosphate-buffered saline. When the MSCs were treated with epigallocatechin gallate, which is purified from green tea catechin, the MSCs showed high viability as well as osteogenic differentiation capability even while suspended in phosphate-buffered saline for 4 days at 4 degrees C. In contrast, nontreated MSCs showed low viability and showed hardly any differentiation. The rate of proliferation of the treated MSCs was much higher than that of the nontreated MSCs. These results indicate the usefulness of epigallocatechin gallate treatment for fabrication of ready-to-use cellular products of MSC suspension.

In vivo osteogenic capability of human mesenchymal cells cultured on hydroxyapatite and on beta-tricalcium phosphate.

The aim of the current study was to examine in vitro osteogenic capability and in vivo bone formation of mesenchymal stromal cells (MSCs) on two kinds of calcium phosphate ceramics. MSCs derived from human bone marrow were seeded on either hydroxyapatite (HA) ceramic or beta-tricalcium phosphate (beta-TCP) ceramic and then cultured in a medium supplemented with a donor's serum, vitamin C, beta-glycerophosphate, and dexamethasone. The culture revealed the expression of alkaline phosphatase activity, indicating the osteogenic differentiation of the MSCs on the ceramics (fabrication of tissue-engineered construct). The constructs were then implanted subcutaneously into nude rats for 8 weeks. New bone formation was observed in both types of ceramics, and human-specific Alu sequence was detected by in situ hybridization analysis. Quantitative microcomputed tomography showed that the volume of the new bone in the HA ceramic was greater than that in the beta-TCP ceramic in six of seven cases. These results suggest that human MSCs cultured on ceramics could retain their osteogenic capability even after ectopic implantation and provide a rationale for the use of tissue-engineered constructs derived from a patient's MSCs and calcium phosphate ceramics in bone tissue regeneration.

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.

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.

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.

[Bone tissue regeneration using stem cells].

In vitro modification of mesenchymal stem cells and materials via tissue engineering is useful for bone regeneration. In this paper, we briefly summarize our bone tissue engineering approach. It is also known that RNA interference (RNAi) can regulate cell proliferation as well as differentiation cascade. Recently, our study using siRNA demonstrated that bone matrix formation by osteoblastic cells can be inhibited by certain types of siRNA. These findings can show that the proliferation/differentiation of the cells used in regenerative medicine could be regulated by siRNA.

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.

High-efficiency DNA injection into a single human mesenchymal stem cell using a nanoneedle and atomic force microscopy.

We describe a low-invasive gene delivery method that uses an etched atomic force microscopy (AFM) tip or nanoneedle that can be inserted into a cell nucleus without causing cellular damage. The nanoneedle is 200 nm in diameter and 6 mum in length and is operated using an AFM system. The probabilities of insertion of the nanoneedle into human mesenchymal stem cells (MSCs) and human embryonic kidney cells (HEK293) were higher than those of typical microinjection capillaries. A plasmid containing the green fluorescent protein (GFP) gene was adsorbed on a poly-L-lysine-modified nanoneedle surface, which was then inserted into primary cultured single human MSCs. A highly efficient gene delivery of over 70% was achieved in human MSCs, which compared very favorably with other major nonviral gene delivery methods (lipofection approximately 50%, microinjection approximately 10 %). The single cells expressing GFP were collected and the amount of delivered DNA in each cell was analyzed. The highest rate of expressed GFP per delivered DNA was achieved using the nanoneedle, because the nanoneedle could be inserted into the nucleus directly without causing significant cell damage.

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.

Tissue-engineered approach for the treatment of steroid-induced osteonecrosis of the femoral head: transplantation of autologous mesenchymal stem cells cultured with beta-tricalcium phosphate ceramics and free vascularized fibula.

Autologous mesenchymal stem cells (MSCs) cultured with beta-tricalcium phosphate (beta-TCP) ceramics and with a free vascularized fibula were transplanted into three patients with steroid-induced osteonecrosis of the femoral head. The average follow-up period was 34 months and the average patient age at the time of surgery was 28 years old. Fifteen milliliters of bone marrow was obtained from the patients 4 weeks before surgery, and was used for in vitro proliferation of MSCs. beta-TCP granules were immersed in the MSC suspension and the cells were further cultured for 2 weeks. Cultured MSCs/beta-TCP composite granules were implanted into the cavity that remained after curettage of necrotic bone; and finally, a free vascularized fibula was grafted. All hips showed preoperative collapse and radiographic progression was observed in two hips postoperatively. Osteonecrosis did not progress any further and early bone regeneration was observed. This tissue-engineered approach has potentials for the treatment of osteonecrosis. However, our results suggested that the present procedure could not be used for cases with severe preoperative collapse.

Assessment of viability and osteogenic ability of human mesenchymal stem cells after being stored in suspension for clinical transplantation.

Human mesenchymal stem cells (MSCs) were suspended in phosphate-buffered saline (PBS) and stored up to 24 h at 4 degrees C, 24 degrees C, and 37 degrees C. More than 80% viability was maintained at any temperature for at least 1 h, then gradually decreased over time. After 24 h, the viabilities at 4 degrees C, 24 degrees C, and 37 degrees C were about 81%, 70%, and 62%, respectively. The MSCs suspended/stored in PBS at 4 degrees C for 24 h also exhibited in vitro osteogenic differentiation capability as evidenced by mineralized matrix formation as well as high alkaline phosphatase activity when cultured in an osteogenic medium. Furthermore, in vivo implantation experiments using the MSCs also demonstrated new bone formation. Because MSCs are known to possess multipotential stem cell characteristics, these data indicate that human MSCs stored in PBS at 4 degrees C could be delivered to distant medical facilities for the purpose of hard tissue and other types of tissue regeneration therapy.

Osteogenic differentiation of human dental papilla mesenchymal cells.

We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.

Tissue engineering approach to the treatment of bone tumors: three cases of cultured bone grafts derived from patients' mesenchymal stem cells.

A novel approach to the treatment of bone tumors using tissue-engineered implants is reported in this study. The number of mesenchymal stem cells (MSCs) obtained from each patient's bone marrow cells was first increased, and the MSCs were forced to differentiate into osteoblasts followed by bone matrix formation on hydroxyapatite (HA) ceramics. The strong osteogenic ability of the implants, as evidenced by high osteoblastic activity, was confirmed. Consequently, the HA surface was covered with the patient's derived cultured osteoblast/bone matrix. The tissue-engineered HA was used to fill the patient's bone cavity after tumor curettage. Immediate healing potential was found by serial plain radiographs and computed tomograhy images, and no adverse reactions were noted in these patients. The results indicate that tissue-engineered osteogenic ceramics might be an alternative to autologous bone grafts.

Observation and quantitative analysis of rat bone marrow stromal cells cultured in vitro on newly formed transparent beta-tricalcium phosphate.

To observe living cell morphology on ceramics by light microscopy, we fabricated a new material-transparent beta - tricalcium phosphate (t-beta TCP) ceramic-for the purpose of serving as a tissue culture substrate. Bone marrow stromal cells (BMSCs) were obtained from rat femora and cultured on both t-beta TCP ceramic disks and culture grade polystyrene (PS) dishes in an osteogenic medium. After 1 day of culture, cell attachment and spreading on both the t-beta TCP and PS substrata were equally and clearly detected by ordinary light microscopy. After 14 days of culture, extensive cell growth, alkaline phosphatase (ALP) staining, and bone mineral deposition could be detected on both substrata. In addition, quantitative biochemical analyses revealed high DNA content, ALP activity, and osteocalcin content of these cultures. This experiment is significant in that all of the results were similarly observed on both the t-beta TCP and PS substrata, indicating the excellent properties of beta TCP ceramics for BMSCs culture towards osteogenic differentiation.

Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells.

Human bone marrow-derived mesenchymal cells contain mesenchymal stem cells (MSCs), which are well known for their osteo/chondrogenic potential and can be used for bone reconstruction. This article reports the viability of cryopreserved human mesenchymal cells and a comparison of the osteogenic potential between noncryopreserved and cryopreserved human mesenchymal cells with MSC-like characteristics, derived from the bone marrow of 28 subjects. The viability of cryopreserved mesenchymal cells was approximately 90% regardless of the storage term (0.3 to 37 months). It is clear by fluorescence-activated cell sorter analysis that the cell surface antigens of both noncryopreserved and cryopreserved mesenchymal cells were negative for hematopoietic cell markers such as CD14, CD34, CD45, and HLA-DR but positive for mesenchymal characteristics such as CD29 and CD105. To monitor the osteogenic potential of the cells, such as alkaline phosphatase (ALP) activity and in vitro mineralization, a subculture was conducted in the presence of dexamethasone, ascorbic acid, and glycerophosphate. No difference in osteogenic potential was found between cells with or without cryopreservation treatment. In addition, cells undergoing long-term cryopreservation (about 3 years) maintained high osteogenic potential. In conclusion, cryopreserved as well as noncryopreserved human mesenchymal cells could be applied for bone regeneration in orthopedics.