Temporal changes guided by mesenchymal stem cells on a 3D microgel platform enhance angiogenesis in vivo at a low-cell dose.

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).

TGF-ß1-Licensed Murine MSCs Show Superior Therapeutic Efficacy in Modulating Corneal Allograft Immune Rejection In Vivo.

Mesenchymal stromal cells (MSCs) are a promising therapeutic option for multiple immune diseases/disorders; however, efficacy of MSC treatments can vary significantly. We present a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with transforming growth factor-ß1 (TGF-ß MSCs) significantly improved their ability to modulate both the phenotype and secretome of inflammatory bone marrow-derived macrophages and significantly increased the numbers of regulatory T lymphocytes following co-culture assays. These TGF-ß MSC-expanded regulatory T lymphocytes also expressed significantly higher levels of PD-L1 and CD73, indicating enhanced suppressive potential. Detailed analysis of T lymphocyte co-cultures revealed modulation of secreted factors, most notably elevated prostaglandin E2 (PGE2). Furthermore, TGF-ß MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for unlicensed MSC-treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (1) therapeutic efficacy of TGF-ß MSCs is Smad2/3-dependent, (2) the enhanced immunosuppressive capacity of TGF-ß MSCs is contact-dependent, and (3) enhanced secretion of PGE2 (via prostaglandin EP4 [E-type prostanoid 4] receptor) by TGF-ß MSCs is the predominant mediator of Treg expansion and T cell activation and is associated with corneal allograft survival. Collectively, we provide compelling evidence for the use of TGF-ß1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity.

Anti-donor antibody induction following intramuscular injections of allogeneic mesenchymal stromal cells.

Allogeneic mesenchymal stromal cells (allo-MSC) are a promising "off-the-shelf" therapy with anti-inflammatory and pro-repair properties. This study investigated humoral immune responses to intramuscular (IM) injections of allo-MSC. Total and isotype-specific anti-donor IgG and donor-specific complement-mediated lysis were determined in sera from healthy mice 2 weeks after single or repeated IM injections of fully mismatched-MHC allo-MSC with comparison to mice receiving syngeneic MSC, allogeneic splenocytes or saline. In mice subjected to hind limb ischemia (HLI), anti-donor IgG was analyzed following IM allo-MSC injection with and without administration of the T-cell immunosuppressant tacrolimus. Recipients of single and repeated IM allo-MSC developed readily-detectable anti-donor IgG. Serum anti-donor IgG levels were similar to those of allo-splenocyte recipients but had higher IgG1/IgG2a ratio and variable capacity for complement-mediated lysis of donor cells. The induced anti-donor IgG bound readily to allo-MSC and this binding was increased following allo-MSC pretreatment with interferon gamma. In mice with HLI, IM injection of allo-MSC into the ischemic limb was also associated with induction of anti-donor IgG but this was abrogated by tacrolimus (FK-506). The results indicate that allo-MSC are inherently immunogenic when delivered intramuscularly to healthy and ischemic mouse hind limb, but induce an IgG1-skewed humoral response that is suppressed by tacrolimus.

Chondrogenic differentiation increases antidonor immune response to allogeneic mesenchymal stem cell transplantation.

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3(+) and CD68(+) infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.

Subconjunctival administration of low-dose murine allogeneic mesenchymal stromal cells promotes corneal allograft survival in mice.

BACKGROUND: Systemic administration of mesenchymal stromal cells (MSCs) has been efficacious in many inflammatory disease settings; however, little data are available on the potential immunomodulatory effects following local MSC administration in the context of corneal transplantation. The purpose of this study was to assess the potential of subconjunctival injection of MSCs to promote corneal allograft survival. METHODS: MSCs were isolated from female C57BL/6 (H-2k) or Balb/c (H-2d) mice and extensively characterized. An allogeneic mouse corneal transplant model was used with Balb/c mice as recipients of C57BL/6 grafts. A dose-finding study starting with 5 × 105 MSCs injected subconjunctivally at day - 7 was tested first followed by a more clinically translatable low-dose single or dual injection strategy on day - 1 and day + 1 before/after transplantation. Graft transparency served as the primary indicator of transplant rejection while neovascularization was also recorded. Lymphocytes (from draining lymph nodes) and splenocytes were isolated from treatment groups on day 2 post-transplantation and characterized by flow cytometry and qRT-PCR. RESULTS: Both high- and low-dose injection of allogeneic MSCs on day - 7 led to 100% graft survival over the observation period. Moreover, low-dose dual subconjunctival injection of 5 × 104 allogeneic MSCs on day - 1 or day + 1 led to 100% allograft survival in transplant recipients (n = 7). We also demonstrate that single administration of allogeneic MSCs on either day - 1 or day + 1 promotes rejection-free graft survival in 100% (n = 8) and 86% (n = 7) of transplanted mice, respectively. Early time point ex vivo analysis suggests modulation of innate immune responses towards anti-inflammatory, pro-repair responses by local MSC administration. CONCLUSION: This work demonstrates that low-dose subconjunctival injection of allogeneic MSCs successfully promotes corneal allograft survival and may contribute to refining future MSC immunotherapies for prevention of corneal allograft rejection.

[Odontoblast-like cell phenotype differentiation of cranial neural crest stem cell in vivo].

OBJECTIVE: To investigate the feasibility of tooth regeneration by seeding cranial neural crest stem cell (CNCSC) in vivo. METHODS: Cranial neural tubes, dissected from mouse E9 d, were explanted onto fibronectin-coated dishes. CNCSC emigrated from the explanted neural tubes, and were cultured in a free-serum medium containing modified DMEM/F12. CNCSC, induced by FGF8, BMP2, TGFbeta1 and dentin matrix non-collagen protein (DMNCP), were cultured with collagen/chitosan, and implanted into the subcutaneous part of immunodeficiency mouse. The expression of collagen I/dentin sialophosphoprotein (DSPP) was analyzed by immunocytochemistry. RESULTS: With the scaffolds destroying, columnar cells possessing polarized nuclei and matrix produced by cells were showed in some regions. Immunohistochemical staining demonstrated that collagen type I and DSPP were expressed throughout the cytoplasm and matrix produced by cells. CONCLUSION: By tissue engineering approach, our experiments further verify the odontoblast-like cell phenotype differentiation of CNCSC in vivo.

Ectopic and in situ bone formation of adipose tissue-derived stromal cells in biphasic calcium phosphate nanocomposite.

Adipose-derived stromal cells (ASCs) have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. A novel biodegradable biphasic calcium phosphate nanocomposite (NanoBCP) comprising beta-tricalcium phosphate matrix and hydroxyl apatite nanofibers is favorable for bone tissue engineering. In this study, ASCs were harvested from Sprague-Dawley (SD) rats and induced to osteogenesis before seeded into porous NanoBCP scaffold. To determine ectopic in vivo osteogenic differentiation, these constructs were implanted in nude mice subcutaneously. Meanwhile, the ability of engineered constructs to stimulate in situ bone repair was assessed in rat critical-size cranial defects. The defects were filled with NanoBCP containing osteogenic ASCs in experimental group; with cell-free NanoBCP in negative controls; and with nothing in blank controls. The retrieved specimens were analyzed with morphological, histological, and molecular methods. Histological analysis of the retrieved specimens from nude mice in experimental group showed obvious ectopic bone formation. There were positive expression of osteopontin (OPN) and osteocalcin (OCN) at RNA and protein level. As for the cranial defects, there was complete repair in experimental group, but only partial repair in negative controls. The radiographs, H&E staining, and Masson's trichrome method showed better bone regeneration at experimental sites. Combining osteogenic ASCs with NanoBCP can lead to formation of ectopic new bone. Furthermore, the approach can also stimulate bone regeneration and repair for the large size bone defects.

Proliferation and pluripotency potential of ectomesenchymal cells derived from first branchial arch.

Cranial neural crest-derived ectomesenchymal cells are multipotential progenitors that contribute to various tissue types during embryogenesis. Their potential to be expanded in culture as a monolayer and to be induced into different cell lineages in vitro has not been previously reported in detail. In this study, the ectomesenchymal cells in the first branchial arch were enzymatically isolated from the mandibular processes of BALB/c mice and were maintained in an intact state in a medium containing leukaemia inhibitory factor. Here, we first evaluated the proliferative activity of the cells after the third passage, using bromodeoxyuridine labelling and in situ hybridization of telomerase mRNA. Positive staining for expression of HNK-1, S-100 and vimentin confirmed that the population of stem cells originated from the ectomesenchyme, which did not express cytokeratin. Then we investigated the molecular and cellular characteristics of the ectomesenchymal cells during their differentiation towards neurogenic, endothelial, myogenic and odontogenic lineages. Expression of multiple lineage-specific genes and proteins was detected by utilizing a range of molecular and biochemical approaches when the cells were transferred to inductive medium. Histological and immunohistochemical analysis of the induced cells at various intervals indicated obvious phenotypic alteration and presence of specific proteins for the differentiated lineages, for example nestin, factor VIII, alpha-SMA and dentin sialophosphoprotein (DSPP), respectively. Correlatively, results of reverse transcription-PCR corroborated at mRNA level the expression of the characteristic molecules during differentiation. Therefore, it is suggested that the ectomesenchymal cells derived from the first branchial arch may represent a novel source of multipotential stem cells capable of undergoing expansion and variant differentiation in vitro.

[A study on transfection of green fluorescence protein gene into human adipose stromal cells in vitro].

OBJECTIVE: To make a comparison on the efficiency of two methods for transfecting Green Fluorescence Protein gene into human adipose tissue-derived stromal cells, and to study the biological properties and multipotential differentiation of gene-transfected cells. METHODS: The human subcutaneous adipose tissue was obtained, digested with one volume of collagenase type I, and then cultured with BGJb medium. After subculture and expansion, the human adipose tissue-derived stromal cells infected with Ad-GFP or liposome were observed and analyzed with fluorescence microscopy and flow cytometry to assess transfection efficiency. The growth curve of transfected adipose tissue-derived stromal cells was protracted. The adipose tissue-derived storomal cells were induced to differentiate into osteoblasts, and non-transfected cells were set as control. RESULTS: 42.5% +/- 1.5 of the human adipose tissue-derived stomal cells infected with Ad-GFP were found to express GFP at a level higher than that of the control of liposome (11.40%). Infected adipose tissue-derived stromal cells were noted to form mineralized nodes by the use of Alizarin Red stain. CONCLUSION: The human adipose tissue-derived stromal cells infected with Ad-GFP can express higher level of GFP, and can maintain the ability of proliferation and differentiation as the non-infected human adipose tissue-derived stromal cells do. The infected adipose tissue-derived stromal cells with Ad-GFP can track the change of adipose tissue-derived stromal cells in the study of multipotential differentiation and can serve as cellular vehicles for systemic gene delivery.

[Cultivation and morphological characteristics of rat adipose tissue-derived vascular endothelial cells in vitro].

The subcutaneous adipose tissue from the inguen of four Sprague-Dawley rats was obtained, then digested with one volume of collagenase type I and cultured with BGJb medium. The obtained adipose stromal cells were induced in human endothelial-SFM for 7 d. The cells were observed under inverted microscope every day and identified by transmission electron microscope and immunocytochemical staining with factor VIII antigen. The results showed the induced cells uniformly had characteristic cobblestone morphology of endothelial cells. Factor VIII antigen staining was positive in cytoplasm. Under transmission electron microscope, the cells displayed many finger like microvilli and numerous lysosomes, mitochondria, a few coarse endoplasmic reticulum and Weibel-Palade bodies. The characteristics of the rat adipose tissue-derived endothelial cells were consistent with those of vascular endothelial cells derived from other tissues. It seems that subcutaneous adipose tissue may represent a new alternative source of endogenous vascular endothelial cells.

[The reconstruction of bioengineer active corneal stroma and its biocompatibility study].

This paper aims to explore the biocompatibility of bioengineer active corneal stroma (BACS), as the biological carrier for cornea reconstruction, to provide the basis for future study on clinic application. The cells and immunogenic components of cornea stroma were removed through different extract methods. A complex of functional corneal stroma cells and acellular corneal stroma was used to reconstruct BACS. Their morphological characteristics and ultrastructures were observed with transmission electron microscope. The complex was grafted into interlamellar stromal pockets. Cells were labeled by BrdU to examine the survival and conversion after grafting. The cells could survive and proliferate in acellular corneal stroma. All the nuclei of the corneal stromal cells showed positive labeling with BrdU in the BACS. After 4 weeks, BACS became transparent; after 8 weeks, the bioengineer active cornea stroma was fully reconstructed.

Variability in Endogenous Perfusion Recovery of Immunocompromised Mouse Models of Limb Ischemia.

Immunocompromised hind limb ischemia (HLI) murine models are essential for preclinical evaluation of human cell-based therapy or biomaterial-based interventions. These models are used to generate proof of principle that the approach is effective and also regulatory preclinical data required for translation to the clinic. However, surgical variations in creation of HLI models reported in the literature introduce variability in the pathological manifestation of the model, in consequence affecting therapeutic endpoints. This study aims to compare the extent of vascular regeneration in HLI-induced immunocompromised murine models to obtain a stable and more reproducible injury model for testing. Athymic and Balb/C nude mice underwent HLI surgery with single and double ligation of femoral artery (FA). The recovery from surgery was observed over a period of 2 weeks with respect to ischemia reperfusion using laser Doppler and clinical signs of necrosis and ambulatory impairment. Double ligation of the FA results in a more severe response to ischemia in Balb/C with endogenous perfusion recovery up to 50% ± 10% compared with 75% ± 20% in athymic nude mice. Single iliac artery (IA) and FA lead to creation of mild ischemia compared with femoral artery-vein (FAV) pair ligation in Balb/C. Microcirculatory parameters indicate significantly lower capillary numbers (26 ± 3/mm(2)) and functional capillary density (203 ± 5 cm/cm(2)) in the FAV group. In this study, we demonstrate a reproducible, arterial double ligation in an immunocompromised Balb/C nude mouse model that exhibits characteristic pathological signs of ischemia with impaired endogenous recovery.

[Expression of human VEGF165 gene in rat bone marrow stromal cells in vitro].

OBJECTIVE: The purpose of this study was to evaluate the feasibility of VEGF165 gene transfection into bone marrow stromal cells and make the groundwork of VEGF gene therapy for the revascularization of bone tissue engineering. METHODS: Bone marrow stromal cells (BMSC) were cultured in vitro, and were tested for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. The vector pcDNA3.1-VEGF165 was transfected into bone marrow stroma cells with the method of liposome mediated or Sofast transfectam reagent. The expression level of human VEGF165 in the transformed cells was detected by immunocytochemistry. RESULTS: The percent of the positive cells after VEGF165 gene transfection mediated by liposome and cationic polymer was 11.34 and 11.42 respectively. There was no significant difference in gene transfer efficacy between liposome and cationic polymer mediated gene delivery. While the percent of the number of survival cells after gene transfection was 74.60 and 85.88 respectively, the cytoxity of liposome was a little higher than that of cationic polymer. CONCLUSION: The experiment demonstrated that rhVEGF165 was successfully expressed in BMSC. This result could serve as a basis for the next step of revascularization of bone tissue engineering.

An injectable elastin-based gene delivery platform for dose-dependent modulation of angiogenesis and inflammation for critical limb ischemia.

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 µg) and IL-10 (10 µg)/eNOS (20 µg) and reduced inflammation with IL-10 (10 µg) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia.

A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses in stem cells.

Development of cell delivery platforms have been driven based on an empirical cytoprotective design. While cell-matrix and cell-cell interactions that influence biochemical effects beyond survival has been limited and overshadowed in an effort to incrementally improve biomimicking properties of the tissue-engineered constructs. Here we demonstrate fabrication of a shape controlled 3D type-I collagen-based microgel platform that can be tuned to modulate angiogenic paracrine- 'angiocrine' responses of human mesenchymal stem cells (hMSCs). Furthermore, these microgels were characterized as a 3D cell culture tool to assess optimal biological response as a function of cell-matrix and cell-cell interactions. Finally, optimised hMSC embedded microgels were shown to induce vascular repair and functional improvement in vivo in a mouse model of hind-limb ischemia. The approach described here in designing a tuneable cell delivery platform using naturally occurring extracellular matrix molecules highlights the need for highly customised matrices with an array of self-assembling proteins that dictate specific cell function resembling the native tissue of interest for repair.

A comparison of the efficacy of transplantation of bone marrow-derived mesenchymal stem cells and unrestricted somatic stem cells on outcome after acute myocardial infarction.

INTRODUCTION: A number of questions remain unanswered in the field of cell therapy for acute myocardial infarction, including what is the optimal cell type, and can therapeutic efficacy be enhanced by conditioning regimens. In this study, we sought to address these questions by directly comparing the effect of bone marrow-derived mesenchymal stem cells and unrestricted somatic stem cells delivered 24 hours post-myocardial infarction and by determining if the therapeutic efficacy of unrestricted somatic stem cells could be enhanced by exposing the cells to guiding factors before cell transplantation. METHODS: Unrestricted somatic stem cells were guided by exposure to 50 ng/mL basic fibroblast growth factor, 20 ng/mL hepatocyte growth factor and 20 ng/mL bone morphogenetic protein-2 for 24 hours. Using a Sprague-Dawley rat model of acute myocardial infarction, we transplanted cells by intramyocardial injection 24 hours post-myocardial infarction. Cardiac function was serially measured using echocardiography, and histological analyses of infarct morphology, angiogenesis and apoptosis were obtained. Transcriptomic and proteomic changes were assessed using microarray and real-time quantitative PCR. RESULTS: When assessed 28 days after the myocardial infarction, the delivery of mesenchymal stem cells 24 hours post-myocardial infarction did not improve ejection fraction (P = 0.19), and did not prevent the decline in ejection fraction observed in the absence of cell therapy (P = 0.17). The administration of unrestricted somatic stem cells also did not improve ejection fraction (P = 0.11), but did prevent a further decline in ejection fraction (P = 0.001). Delivery of guided unrestricted somatic stem cells significantly improved ejection fraction (P = 0.03). Guided unrestricted somatic stem cells restored function to a greater extent than mesenchymal stem cells (P = 0.03). The infarct area (P = 0.2), apoptosis (P = 0.07) and angiogenesis (P = 0.09) did not differ between groups. Microarray analysis revealed that, following pre-implantation guiding, the gene groupings of mitosis, signalling and angiogenesis were highly overrepresented, mediators of apoptosis were overrepresented, and cardiomyocyte-associated genes were not differentially expressed. CONCLUSIONS: These results suggest that guided unrestricted somatic stem cells have a moderate capacity to repair cardiac damage and that they are more effective than mesenchymal stem cells in restoring cardiac function after a myocardial infarction. The mechanism of the benefit was not fully elucidated in this study, but these observations may be mediated by favorable dysregulation of angiogenic and apoptotic gene groupings.

Recovery of cardiac function mediated by MSC and interleukin-10 plasmid functionalised scaffold.

Stem cell transplantation has been suggested as a treatment for myocardial infarction, but clinical studies have yet to demonstrate conclusive, positive effects. This may be related to poor survival of the transplanted stem cells due to the inflammatory response following myocardial infarction. To address this, a scaffold-based stem cell delivery system was functionalised with anti-inflammatory plasmids (interleukin-10) to improve stem cell retention and recovery of cardiac function. Myocardial infarction was induced and these functionalised scaffolds were applied over the infarcted myocardium. Four weeks later, stem cell retention, cardiac function, remodelling and inflammation were quantified. Interleukin-10 gene transfer improved stem cell retention by more than five-fold and the hearts treated with scaffold, stem cells and interleukin-10 had significant functional recovery compared to the scaffold control (scaffold: -10 ± 7%, scaffold, interleukin-10 and stem cells: +7 ± 6%). This improved function was associated with increased infarcted wall thickness and increased ratios of collagen type III/type I, decreased cell death, and a change in macrophage markers from mainly cytotoxic in the scaffold group to mainly regulatory in scaffold, stem cells and interleukin-10 group. Thus, treatment of myocardial infarction with stem cells and interleukin-10 gene transfer significantly improved stem cell retention and ultimately improved overall cardiac function.

[Experimental study on cell adhesion characteristic between porous biphasic calcium phosphate nanocomposite and bone marrow mesenchymal stem cells in vitro].

OBJECTIVE: To study the adhesion characteristic in vitro between porous biphasic calcium phosphate (BCP) nanocomposite and bone marrow mesenchymal stem cells (MSCs) that have been induced and proliferated. METHODS: MSCs obtained from SD rat bone marrow were in vitro induced and proliferated. After their osteoblastic phenotype were demonstrated, MSCs were seeded onto prepared porous BCP nanocomposite (experiment group)and common porous hydroxyapatite (control group). Their adhesion situation was analyzed by scanning electron microscope. The initial optimal cell seeding density was investigated between new pattern porous BCP nanocomposite and MSCs by MTT automated colorimetric microassay method. RESULTS: The differentiation of MSCs to osteoblastic phenotype were demonstrated by the positive staining of mineralized node, alkaline phosphatase (ALP) and collagen type I, the most appropriate seeding density between them was 2 X 10(6)/ml. The maximal number which MSCs could adhere to porous BCP nanocomposite was 1.28 X 10(7)/cm3. CONCLUSION: MSCs can differentiate to osteoblastic phenotype. The MSCs were well adhered to porous BCP nanocomposite.

[A study on repairing mandibular defect by means of tissue-engineering and human bone morphogenetic protein-2 gene transfection in osteoporotic rats].

OBJECTIVE: To investigate the feasibility of repairing bone defect with methods of tissue-engineering and human bone morphogenetic protein-2 (hBMP-2) gene transfection in osteoporotic rats. METHODS: Twenty-four 6-month-old female Sprague-Dawley rats underwent ovariectomy, while 8 rats received sham-operations. Three months later, bone mesenchymal stem cells (BMSC) harvested from osteoporotic rats were divided into two groups randomly. Experimental group were transfected by recombinant plasmid carrying hBMP-2 gene, and control group left untreated. All BMSC were seeded into coralhydroxyapatite scaffolds. Then the cell/scaffold constructs were implanted into the defect site created in the ramus of mandible of osteoporotic rats respectively. RESULTS: Positive results were confirmed by immunohistochemistry and in situ hybridization in experimental group. New bone formation was found at the margin of the defect treated with the BMSC modified by hBMP-2 gene transfer at 4 weeks after implantation and appeared mature 8 weeks after the treatment. However, the amount of newly formed bone was much less and there was some adipose tissue at defect margins 8 weeks after implantation in control group. CONCLUSIONS: The results of this experiment indicate that BMSC-mediated rhBMP-2 gene therapy in conjunction with bone tissue engineering may allow for successful treatment of large bone defects in osteoporosis rats.

Induction of transforming growth factor-beta 1 on dentine pulp cells in different culture patterns.

Recent studies have documented that TGF-beta1 takes part in dental pulp tissue repair. Moreover, dental pulp cells have the potential to differentiate into odontoblast-like cells and produce reparative dentine in this process. However, the molecular mechanisms and potential interactions between TGF-beta1 and dental pulp cells are not clear due to the complexity of the pulp/dentine microenvironment. In this study, we investigated the induction of TGF-beta1 on the dental pulp cells in cell culture, tissue culture and three-dimensional culture patterns. These results demonstrated that TGF-beta1 significantly increased the proliferation of cells and activity of ALPase. Dental pulp cells cultured in the presence of TGF-beta1 formed mineralization nodules. In the organ culture, dental pulp cells treated with TGF-beta1 differentiated into odontoblast-like cells and formed a pulp-dentinal complex; and TGF-beta1 significantly induced synthesis of dentine relative proteins DSPP, DMP-1. The dental pulp cells share some characteristics of the odontoblast, such as a parallel arrangement with columnar form and a unilateral cell process. Together, these data indicate that TGF-beta1 can make dental pulp cells differentiated into odontoblast-like cells and form the pulp-dentinal complex. Moreover, these results suggest that TGF-beta1 is an important regulatory factor in odontoblast differentiation during tooth development and pulp repair.