自组装多肽水凝胶支架中MSCs的三维培养及成骨分化

使用一种新型人工设计自组装多肽(RADA16)水凝胶作为三维培养支架评价MSCs成骨分化情况。将人骨髓MSCs培养增殖后接种到水凝胶中,在成骨分化培养液中进一步培养1～3周。荧光染色法观察细胞形态和存活情况;组织学染色检测MSCs ALP活性;半定量RT-PCR分析成骨特异性基因的表达。绝大多数MSCs在水凝胶支架内能够存活,呈纺锤样形态。诱导培养后蛋白和基因表达水平均检测到ALP活性,在14天时达到峰值。骨晚期分化特异性基因BSP也有表达,且表达量随培养时间延长而增多。自组装多肽水凝胶为MSCs的黏附生长及向成骨细胞分化提供良好的三维微环境,有望成为极具吸引力的骨组织工程支架材料。     

Effect of Inducible BMP-7 Expression on the Osteogenic Differentiation of Human Dental Pulp Stem Cells

BMP-7 has shown inductive potential for in vitro osteogenic differentiation of mesenchymal stem cells, which are an ideal resource for regenerative medicine. Externally applied, recombinant BMP-7 was able to induce the osteogenic differentiation of DPSCs but based on our previous results with BMP-2, we aimed to study the effect of the tetracyclin-inducible BMP-7 expression on these cells. DPSC, mock, and DPSC-BMP-7 cell lines were cultured in the presence or absence of doxycycline, then alkaline phosphatase (ALP) activity, mineralization, and mRNA levels of different osteogenic marker genes were measured. In the DPSC-BMP-7 cell line, the level of BMP-7 mRNA significantly increased in the media supplemented with doxycycline, however, the expression of Runx2 and noggin genes was upregulated only after 21 days of incubation in the osteogenic medium with doxycycline. Moreover, while the examination of ALP activity showed reduced activity in the control medium containing doxycycline, the accumulation of minerals remained unchanged in the cultures. We have found that the induced BMP-7 expression failed to induce osteogenic differentiation of DPSCs. We propose three different mechanisms that may worth investigating for the engineering of expression systems that can be used for the induction of differentiation of mesenchymal stem cells.     

Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.     

Development of Electrospun Composite Fibers in Multiscale Structure and Investigating the Performance on Proliferation and Osteogenic Differentiation of ADSCs

Electrospun composite membranes in multiscale structures are developed for bone tissue engineering. Aligned polycaprolactone (PCL) fibers entrapping CA‐HAp microparticles (containing CaCO₃, hydroxyapatite, and casein in a hierarchical organization) are electrospun to find whether synergistic effects of fiber alignment and CA‐HAp microparticles on improving osteogenic differentiation can be obtained. CA‐HAp microparticles are in a spherical morphology of 1.42 ± 0.26 µm. Their presence increases fiber diameter and does not significantly affect fiber alignment. On all membranes, adipose derived stem cells (ADSCs) from humans spread very well. On a random group, cells distribute randomly and the presence of CA‐HAp microparticles facilitates cell proliferation, especially for the one at CA‐HAp/PCL 50 wt%; the one at CA‐HAp/PCL 20 wt% shows significantly much higher alkaline phosphatase (ALP) activity (112.0% higher) than the pure PCL membrane. On aligned samples, cells align along fibers and expression of ALP is enhanced. However, at the same composition (CA‐HAp/PCL 20 wt%), the random sample has much higher ALP activity than the aligned sample. The expressions of osteogenic marker genes are also evaluated. Combining the results and the applicability of membranes together, the random membrane at CA‐HAp/PCL 20 wt% is the best candidate for bone tissue engineering.     

Comparison of Osteogenic Potentials of Dental Pulp and Bone Marrow Mesenchymal Stem Cells Using the New Cell Transplantation Platform,CellSaic, in a Rat Congenital Cleft-Jaw Model

Scaffolds stimulate cell proliferation and differentiation and play major roles in providing growth and nutrition factors in the repair of bone defects. We used the recombinant peptide Cellnest™ to prepare the three-dimensional stem cell complex, CellSaic, and evaluated whether CellSaic containing rat dental pulp stem cells (rDPSCs) was better than that containing rat bone marrow stem cells (rBMSCs). rDPSC-CellSaic or rBMSC-CellSaic, cultured with or without osteogenic induction medium, formed the experimental and control groups, respectively. Osteoblast differentiation was evaluated in vitro and transplanted into a rat model with a congenital jaw fracture. Specimens were collected and evaluated by microradiology and histological analysis. In the experimental group, the amount of calcium deposits, expression levels of bone-related genes (RUNX2, ALP, BSP, and COL1), and volume of mineralized tissue, were significantly higher than those in the control group (p < 0.05). Both differentiated and undifferentiated rDPSC-CellSaic and only the differentiated rBMSC-CellSaic could induce the formation of new bone tissue. Overall, rBMSC-CellSaic and rDPSC-CellSaic made with Cellnest™ as a scaffold, provide excellent support for promoting bone regeneration in rat mandibular congenital defects. Additionally, rDPSC-CellSaic seems a better source for craniofacial bone defect repair than rBMSC-CellSaic, suggesting the possibility of using DPSCs in bone tissue regenerative therapy.     

黄芪总黄酮对大鼠原代成骨细胞的影响及其机制研究

在大鼠颅骨原代成骨细胞中加入不同浓度的黄芪总黄酮(ATF),用MTT法检测细胞增殖,用碱性磷酸酶(ALP)活性、Ⅰ型胶原及骨钙素水平检测细胞分化,用茜素红染色法检测细胞矿化,用ELISA法和Western blot方法检测成骨细胞中骨形成蛋白(BMP-2)及核心结合因子(Runx-2)的表达。结果表明,ATF能剂量依赖性地促进大鼠原代成骨细胞的增殖、分化及矿化,同时上调成骨细胞中BMP-2和Runx-2蛋白的表达。提示ATF可能通过上调成骨细胞中BMP-2和Runx-2的表达来促进成骨细胞的成骨活性。     

Osteogenic Potential of Mesenchymal Stem Cells from Adipose Tissue,Bone Marrow and Hair Follicle Outer Root Sheath in a 3D Crosslinked Gelatin-Based Hydrogel

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.     

Enamel Matrix Derivative Promote Primary Human Pulp Cell Differentiation and Mineralization

Enamel matrix derivative (EMD) has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5–50 μg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10⁻⁸ M dexamethasone (DEX) for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 μg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel), and the dentinogenic markers dentin sialophosphoprotein (DSSP) and dentin matrix acidic phosphoprotein 1 (DMP1), as well as the osteogenic markers osteocalcin (OC, BGLAP) and collagen type 1 (COL1A1). Whereas, only EMD had effect on alkaline phosphatase (ALP) mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant proteins (MCP-1) in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation.     

Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.     

Artificial Extracellular Matrices Containing Bioactive Glass Nanoparticles Promote Osteogenic Differentiation in Human Mesenchymal Stem Cells

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect.     

Modeling In Vitro Osteoarthritis Phenotypes in a Vascularized Bone Model Based on a Bone-Marrow Derived Mesenchymal Cell Line and Endothelial Cells

The subchondral bone and its associated vasculature play an important role in the onset of osteoarthritis (OA). Integration of different aspects of the OA environment into multi-cellular and complex human, in vitro models is therefore needed to properly represent the pathology. In this study, we exploited a mesenchymal stromal cell line/endothelial cell co-culture to produce an in vitro human model of vascularized osteogenic tissue. A cocktail of inflammatory cytokines, or conditioned medium from mechanically-induced OA engineered microcartilage, was administered to this vascularized bone model to mimic the inflamed OA environment, hypothesizing that these treatments could induce the onset of specific pathological traits. Exposure to the inflammatory factors led to increased network formation by endothelial cells, reminiscent of the abnormal angiogenesis found in OA subchondral bone, demineralization of the constructs, and increased collagen production, signs of OA related bone sclerosis. Furthermore, inflammation led to augmented expression of osteogenic (alkaline phosphatase (ALP) and osteocalcin (OCN)) and angiogenic (vascular endothelial growth factor (VEGF)) genes. The treatment, with a conditioned medium from the mechanically-induced OA engineered microcartilage, also caused increased demineralization and expression of ALP, OCN, ADAMTS5, and VEGF; however, changes in network formation by endothelial cells were not observed in this second case, suggesting a possible different mechanism of action in inducing OA-like phenotypes. We propose that this vascularized bone model could represent a first step for the in vitro study of bone changes under OA mimicking conditions and possibly serve as a tool in testing anti-OA drugs.     

An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast

The paper explored the regulatory role of oligodeoxynucleotides (ODNs) with specific sequences in the proliferation and activation of osteoblast, using human osteoblast-like cell line MG 63 as the model. Through the administration of ODNs to MG 63 cells at a concentration of 1.0 μg/mL, ODN MT01 with positive effects on proliferation and activation of osteoblast was selected from 11 different ODNs by methyl thiazolyl tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity measurement. To get a deeper insight into the molecular mechanism, effects of ODN MT01 treatment on the expression level of Sp7, runx-2, collagen-I, osteoprotegerin (OPG) and RANK ligand (RANKL) were determined using quantitative real time PCR and Western blotting. Remarkably, the mRNA and protein expression levels of Sp7, runx-2, collagen-I and OPG were improved after ODN MT01 treatment. Meanwhile, the protein expression level of RANKL was dramatically decreased. These results suggested that ODN MT01 had a significant impact in facilitating osteogenic proliferation and activation, and provided a direct evidence for the notion that single strand ODN could regulate the balance of bone formation and resorption, and thus was of great potential in the rebuilding of alveolar bone.     

Minocycline Loaded Hybrid Composites Nanoparticles for Mesenchymal Stem Cells Differentiation into Osteogenesis

Bone transplants are used to treat fractures and increase new tissue development in bone tissue engineering. Grafting of massive implantations showing slow curing rate and results in cell death for poor vascularization. The potentials of biocomposite scaffolds to mimic extracellular matrix (ECM) and including new biomaterials could produce a better substitute for new bone tissue formation. A purpose of this study is to analyze polycaprolactone/silk fibroin/hyaluronic acid/minocycline hydrochloride (PCL/SF/HA/MH) nanoparticles initiate human mesenchymal stem cells (MSCs) proliferation and differentiation into osteogenesis. Electrospraying technique was used to develop PCL, PCL/SF, PCL/SF/HA and PCL/SF/HA/MH hybrid biocomposite nanoparticles and characterization was analyzed by field emission scanning electron microscope (FESEM), contact angle and Fourier transform infrared spectroscopy (FT-IR). The obtained results proved that the particle diameter and water contact angle obtained around 0.54 ± 0.12 to 3.2 ± 0.18 µm and 43.93 ± 10.8° to 133.1 ± 12.4° respectively. The cell proliferation and cell-nanoparticle interactions analyzed using (3-(4,5-dimethyl thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) MTS assay (Promega, Madison, WI, USA), FESEM for cell morphology and 5-Chloromethylfluorescein diacetate (CMFDA) dye for imaging live cells. Osteogenic differentiation was proved by expression of osteocalcin, alkaline phosphatase activity (ALP) and mineralization was confirmed by using alizarin red (ARS). The quantity of cells was considerably increased in PCL/SF/HA/MH nanoparticles when compare to all other biocomposite nanoparticles and the cell interaction was observed more on PCL/SF/HA/MH nanoparticles. The electrosprayed PCL/SF/HA/MH biocomposite nanoparticle significantly initiated increased cell proliferation, osteogenic differentiation and mineralization, which provide huge potential for bone tissue engineering.     

Antiosteoporotic Nanohydroxyapatite Zoledronate Scaffold Seeded with Bone Marrow Mesenchymal Stromal Cells for Bone Regeneration: A 3D In Vitro Model

Background: Bisphosphonates are widely employed drugs for the treatment of pathologies with high bone resorption, such as osteoporosis, and display a great affinity for calcium ions and apatitic substrates. Here, we aimed to investigate the potentiality of zoledronate functionalized hydroxyapatite nanocrystals (HAZOL) to promote bone regeneration by stimulating adhesion, viability, metabolic activity and osteogenic commitment of human bone marrow derived mesenchymal stromal cells (hMSCs). Methods: we adopted an advanced three-dimensional (3D) in vitro fracture healing model to study porous scaffolds: hMSCs were seeded onto the scaffolds that, after three days, were cut in halves and unseeded scaffolds were placed between the two halves. Scaffold characterization by X-ray diffraction, transmission and scanning electron microscopy analyses and cell morphology, viability, osteogenic differentiation and extracellular matrix deposition were evaluated after 3, 7 and 10 days of culture. Results: Electron microscopy showed a porous and interconnected structure and a uniform cell layer spread onto scaffolds. Scaffolds were able to support cell growth and cells progressively colonized the whole inserts in absence of cytotoxic effects. Osteogenic commitment and gene expression of hMSCs were enhanced with higher expressions of ALPL, COL1A1, BGLAP, RUNX2 and Osterix genes. Conclusion: Although some limitations affect the present study (e.g., the lack of longer experimental times, of mechanical stimulus or pathological microenvironment), the obtained results with the adopted experimental setup suggested that zoledronate functionalized scaffolds (GHAZOL) might sustain not only cell proliferation, but positively influence osteogenic differentiation and activity if employed in bone fracture healing.     

Dual Delivery of BMP-2 and bFGF from a New Nano-Composite Scaffold,Loaded with Vascular Stents for Large-Size Mandibular Defect Regeneration

The aim of this study was to investigate the feasibility and advantages of the dual delivery of bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) from nano-composite scaffolds (PLGA/PCL/nHA) loaded with vascular stents (PLCL/Col/nHA) for large bone defect regeneration in rabbit mandibles. Thirty-six large bone defects were repaired in rabbits using engineering bone composed of allogeneic bone marrow mesenchymal stem cells (BMSCs), bFGF, BMP-2 and scaffolds composed of PLGA/PCL/nHA loaded with PLCL/Col/nHA. The experiments were divided into six groups: BMSCs/bFGF/BMP-2/scaffold, BMSCs/BMP-2/scaffold, BMSCs/bFGF/scaffold, BMSCs/scaffold, scaffold alone and no treatment. Sodium alginate hydrogel was used as the carrier for BMP-2 and bFGF and its features, including gelling, degradation and controlled release properties, was detected by the determination of gelation and degradation time coupled with a controlled release study of bovine serum albumin (BSA). AlamarBlue assay and alkaline phosphatase (ALP) activity were used to evaluate the proliferation and osteogenic differentiation of BMSCs in different groups. X-ray and histological examinations of the samples were performed after 4 and 12 weeks post-implantation to clarify new bone formation in the mandible defects. The results verified that the use of sodium alginate hydrogel as a controlled release carrier has good sustained release ability, and the combined application of bFGF and BMP-2 could significantly promote the proliferation and osteogenic differentiation of BMSCs (p < 0.05 or p < 0.01). In addition, X-ray and histological examinations of the samples exhibited that the dual release group had significantly higher bone formation than the other groups. The above results indicate that the delivery of both growth factors could enhance new bone formation and vascularization compared with delivery of BMP-2 or bFGF alone, and may supply a promising way of repairing large bone defects in bone tissue engineering.     

Matricryptic peptide-inspired hydrogels for promoting osteogenic differentiation

In the natural process of bone repair, matricryptic peptides were activated by up-regulated ECM-degrading enzymes and properly released at bone injury sites, which plays critical roles in bone self-healing. Inspired by this natural process, here, matricryptic peptide-inspired (MPI) hydrogels were designed to promote osteogenic differentiation. MPI hydrogels can be degraded by enzymes and during this time, the masked bioactive components were released to promote osteogenic differentiation. These MPI hydrogels which normally serve as structural support for cell proliferation while promote osteogenic differentiation using embedded osteogenic BFP-1 peptides after degradation may provide a novel design for bone regeneration materials.