Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

BACKGROUND A major problem in the healing of bone defects is insufficient or absent blood supply within the defect.To overcome this challenging problem,a plethora of approaches within bone tissue engineering have been developed recently.Bearing in mind that the interplay of various diffusible factors released by endothelial cells(ECs)and osteoblasts(OBs)have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions,we set the focus on the simultaneous application of these cell types together with platelet-rich plasma(PRP)as a growth factor reservoir within ectopic bone tissue engineering constructs.AIM To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells(ASCs)induced into ECs and OBs.METHODS ASCs isolated from adipose tissue,induced in vitro into ECs,OBs or just expanded were used for implant construction as followed:BPEO,endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix;BPUI,uninduced ASCs with PRP and bone mineral matrix;BC(control),only bone mineral matrix.At 1,2,4 and 8 wk after subcutaneous implantation in mice,implants were extracted and endothelial-related and bone-related gene expression were analyzed,while histological analyses were performed after 2 and 8 wk.RESULTS The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation.BC had the lowest endothelial-related gene expression,weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO.Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO.BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs.Except Vwf,endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk.Tissue regression was noticed in BPEO constructs after 8 wk.CONCLUSION Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis,but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.     

牙周骨再生过程中BMP-1表达变化的临床意义

目的:观察在牙周骨再生过程中骨形态发生蛋白-1(BMP-1)的表达的变化情况及临床意义。方法:以我院收治的牙周骨缺损患者196例作为研究对象,采用常规的基础牙周治疗(龈上洁治彻底,龈下刮治,整平根面)处理后,通过引导组织再生术(GTR)技术对骨缺损进行修复的同时,植入Bio-oss人工骨材料和Bio-gide胶原膜。在治疗前和治疗后,分别测定患者血清BMP-1水平,分析BMP-1表达水平与牙周骨缺损修复的关系。结果:与治疗前相比,患者治疗后的牙周骨缺损均有所修复,所有患者在术后所有时间点的PPD和骨密度值与治疗前相比均显著改善(P0.05)。治疗6个月后,牙周骨密度出现小幅度下降,但显著高于治疗前,差异具有统计学意义(P0.05)。与治疗前相比,所有患者治疗后1 d、2 d、5 d、10 d、1个月、3个月、6个月血清BMP-1的水平均显著升高,差异具有统计学意义(P0.05),且治疗后BMP-1水平的变化与PPD呈显著负相关(P0.05),但与骨密度的相关性不显著(P0.05)。结论:血清BMP-1在牙周骨修复过程存在动态变化与患者牙周骨术后恢复相关。     

Application of autologous periosteal cells for the regeneration of class III furcation defects in Beagle dogs

The aim of this study was to evaluate the healing of class III furcation defects following transplantation of autogenous periosteal cells combined with β-tricalcium phosphate (β-TCP). Periosteal cells obtained from Beagle dogs’ periosteum explant cultures, were inoculated onto the surface of β-TCP. Class III furcation defects were created in the mandibular premolars. Three experimental groups were used to test the defects’ healing: group A, β-TCP seeded with periosteal cells were transplanted into the defects; group B, β-TCP alone was used for defect filling; and group C, the defect was without filling materials. Twelve weeks post surgery, the tissue samples were collected for histology, immunohistology and X-ray examination. It was found that both the length of newly formed periodontal ligament and the area of newly formed alveolar bone in group A, were significantly increased compared with both group B and C. Furthermore, both the proportion of newly formed periodontal ligament and newly formed alveolar bone in group A were much higher than those of group B and C. The quantity of cementum and its percentage in the defects (group A) were also significantly higher than those of group C. These results indicate that autogenous periosteal cells combined with β-TCP application can improve periodontal tissue regeneration in class III furcation defects.     

Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.     

Bone morphogenetic proteins in craniofacial and periodontal tissue engineering: experimental studies in the non-human primate Papio ursinus

The bone morphogenetic and osteogenic proteins (BMPs/OPs), pleiotropic members of the transforming growth factor-beta (TGF-beta) supergene family act as soluble signals for the de novo initiation of bone formation, sculpting the multicellular mineralized structures of the bone-bone marrow organ. The strikingly pleiotropic effects of BMPs/OPs spring from amino acid sequence variations in the carboxy-terminal domain and in the transduction of distinct signalling pathways by individual Smad proteins after transmembrane serine/threonine kinase complexes of type I and II receptors. BMPs/OPs are the common molecular initiators deployed for embryonic development and the induction of bone formation and regeneration in postnatal osteogenesis. Naturally derived BMPs/OPs extracted and purified from baboon and bovine bone matrices induce complete regeneration of non-healing calvarial defects in the non-human primate Papio ursinus as well as the induction of cementogenesis and the morphogenesis of a periodontal ligament system with a faithful insertion of Sharpey's fibers into the newly formed cementum. gamma-Irradiated recombinant human osteogenic protein-1 (hOP-1) delivered by xenogeneic bovine collagenous bone matrices completely regenerated and maintained the architecture of the induced bone after treatment of calvarial defects with single applications of doses of 0.1, 0.5 and 2.5mg hOP-1 per gram of carrier matrix. The long-term implantation of hOP-1 delivered by gamma-irradiated bovine bone matrices induced the regeneration of the three essential components of the periodontium, i.e. cementum, periodontal ligament and alveolar bone. The osteogenic proteins of the TGF-beta superfamily are sculpting tissue constructs that engineer skeletal tissue regeneration in molecular terms. The pleiotropy of the signalling molecules of the TGF-beta superfamily is highlighted by the redundancy of molecular signals initiating bone formation, including the TGF-beta isoforms per se, powerful inducers of endochondral bone formation but in the primate only. The induction of bone develops a mosaic structure in which members of the TGF-beta superfamily singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis.     

Novel application of stem cell-derived factors for periodontal regeneration

The effect of conditioned medium from cultured mesenchymal stem cells (MSC-CM) on periodontal regeneration was evaluated. In vitro, MSC-CM stimulated migration and proliferation of dog MSCs (dMSCs) and dog periodontal ligament cells (dPDLCs). Cytokines such as insulin-like growth factor, vascular endothelial growth factor, transforming growth factor-β1, and hepatocyte growth factor were detected in MSC-CM. In vivo, one-wall critical-size, intrabony periodontal defects were surgically created in the mandible of dogs. Dogs with these defects were divided into three groups that received MSC-CM, PBS, or no implants. Absorbable atelo-collagen sponges (TERUPLUG®) were used as a scaffold material. Based on radiographic and histological observation 4 weeks after transplantation, the defect sites in the MSC-CM group displayed significantly greater alveolar bone and cementum regeneration than the other groups. These findings suggest that MSC-CM enhanced periodontal regeneration due to multiple cytokines contained in MSC-CM.     

Effects of concomitant use of fibroblast growth factor (FGF)-2 with beta-tricalcium phosphate (β-TCP) on the beagle dog 1-wall periodontal defect model

The effects of concomitant use of fibroblast growth factor-2 (FGF-2) and beta-tricalcium phosphate (β-TCP) on periodontal regeneration were investigated in the beagle dog 1-wall periodontal defect model. One-wall periodontal defects were created in the mesial portion of both sides of the mandibular first molars, and 0.3% FGF-2 plus β-TCP or β-TCP alone was administered. Radiographic evaluation was performed at 0, 3, and 6 weeks. At 6 weeks, the periodontium with the defect site was removed and histologically analyzed. Radiographic findings showed that co-administration of FGF-2 significantly increased bone mineral contents of the defect sites compared with β-TCP alone. Histologic analysis revealed that the length of the regenerated periodontal ligament, the cementum, distance to the junctional epithelium, new bone height, and area of newly formed bone were significantly increased in the FGF-2 group. No abnormal inflammatory response or ankylosis was observed in either group. These findings indicate the efficacy of concomitant use of FGF-2 and β-TCP as an osteoconductive material for periodontal regeneration following severe destruction by progressive periodontitis.     

Adipose-derived stem cells alleviate osteoporosis by enchancing osteogenesis and inhibiting adipogenesis in a rabbit model

Background aimsOsteoporosis (OP) is characterized by a reduction in bone quality, which is associated with inadequacies in bone marrow mesenchymal stromal cells (BMSCs). As an alternative cell source to BMSCs, adipose-derived stem cells (ASCs) have been investigated for bone repair because of their osteogenic potential and self-renewal capability. Nevertheless, whether autologous ASCs can be used to promote bone regeneration under osteoporotic conditions has not been elucidated.MethodsThe OP rabbit model was established by means of bilateral ovariectomy (OVX). Both BMSCs and ASCs were harvested from OVX rabbits and expanded in vitro. The effects of osteogenic-induced ASCs on the in vitro adipogenic and osteogenic capabilities of BMSCs were evaluated. Autologous ASCs were then encapsulated by calcium alginate gel and transplanted into the distal femurs of OVX rabbits (n = 12). Hydrogel without loading cells was injected into the contralateral femurs as a control. Animals were killed for investigation at 12 weeks after transplantation.ResultsOsteogenic-induced ASCs were able to promote osteogenesis and inhibit adipogenesis of osteoporotic BMSCs through activation of the bone morphogenetic protein 2/bone morphogenetic protein receptor type IB signal pathway. Local bone mineral density began to increase at 8 weeks after ASC transplantation (P < 0.05). At 12 weeks, micro–computed tomography and histological evaluation revealed more new bone formation in the cell-treated femurs than in the control group (P < 0.05).ConclusionsThis study demonstrated that ASCs could stimulate proliferation and osteogenic differentiation of BMSCs in vitro and enhance bone regeneration in vivo, which suggests that autologous osteogenic-induced ASCs might be useful to alleviate OP temporally.     

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.     

Wnt3a promotes differentiation of human bone marrow-derived mesenchymal stem cells into cementoblast-like cells

Cementum is a calcified, avascular connective tissue that laminates the root of a tooth and plays a pivotal role in the development, homeostasis, and regeneration of a periodontal tissue. As a potential treatment for periodontal tissue defects in the patient with chronic periodontitis, much attention has been paid to tissue engineering combined with mesenchymal stem cells for regenerating periodontal tissues including cementum. However, limited information is available for the molecular factors that have impacts on the differentiation of mesenchymal stem cells into cementoblasts. Here, we focus on the effect of Wnt3a as a potential inducer and tested the effect of this protein in vitro using human bone marrow-derived mesenchymal stem cells. It was found that, when cells were cultured in an osteogenic medium containing Wnt3a, cementoblast-specific genes, such as cementum protein 1 and cementum attachment protein, as well as bone-related genes were significantly upregulated. These results suggest that Wnt3a promotes differentiation of the cells into cementoblast-like cells. Further experiments were carried out using inhibitors to gain deeper insights into molecular mechanisms underlying the observed differentiation. As a result, we conclude that Wnt3a-triggered differentiation into cementoblast-like cells is the consequence of the activation of the canonical Wnt signaling pathway with possible involvement of the non-canonical pathway.     

Comparison of adult stem cells derived from multiple stem cell niches

Objectives

Adult stem cells (ASCs) have great potential for tissue regeneration; however, comparative studies of ASCs from different niches are required to understand the characteristics of each population for their potential therapeutic uses.

Results

We compared the proliferation, stem cell marker expression, and differentiation potential of ASCs from bone marrow, skin dermis, and adipose tissue. ASCs from bone marrow and skin dermis showed 50–100 % increased proliferation in comparison to the ASCs from adipose tissues. Furthermore, ASCs from each stem cell niche showed differential expression of stem cell marker genes, and preferentially differentiated into cell types of their tissue of origin.

Conclusion

Different characters of each ASC might be major factors for their effective use for therapeutics and tissue regeneration.

     

Enhancement of jaw bone regeneration via ERK1/2 activation using dedifferentiated fat cells

Background aimsMesenchymal stem/stromal cells (MSCs) are multipotent and self-renewing cells that are extensively used in tissue engineering. Adipose tissues are known to be the source of two types of MSCs; namely, adipose tissue–derived MSCs (ASCs) and dedifferentiated fat (DFAT) cells. Although ASCs are sometimes transplanted for clinical cytotherapy, the effects of DFAT cell transplantation on mandibular bone healing remain unclear.MethodsThe authors assessed whether DFAT cells have osteogenerative potential compared with ASCs in rats in vitro. In addition, to elucidate the ability of DFAT cells to regenerate the jaw bone, the authors examined the effects of DFAT cells on new bone formation in a mandibular defect model in (i) 30-week-old rats and (ii) ovariectomy-induced osteoporotic rats in vivo.ResultsOsteoblast differentiation with bone morphogenetic protein 2 (BMP-2) or osteogenesis-induced medium upregulated the osteogenesis-related molecules in DFAT cells compared with those in ASCs. BMP-2 activated the phosphorylation signaling pathways of ERK1/2 and Smad2 in DFAT cells, but minor Smad1/5/9 activation was noted in ASCs. The transplantation of DFAT cells into normal or ovariectomy-induced osteoporotic rats with mandibular defects promoted new bone formation compared with that seen with ASCs.ConclusionsDFAT cells promoted osteoblast differentiation and new bone formation through ERK1/2 and Smad2 signaling pathways in vitro. The transplantation of DFAT cells promoted new mandibular bone formation in vivo compared with that seen with ASCs. These results suggest that transplantation of ERK1/2-activated DFAT cells shorten the mandibular bone healing process in cytotherapy.     

Cementum is key to periodontal tissue regeneration: A review on apatite microstructures for creation of novel cementum-based dental implants

The cementum is the outermost layer of hard tissue covering the dentin within the root portion of the teeth. It is the only hard tissue with a specialized structure and function that forms a part of both the teeth and periodontal tissue. As such, cementum is believed to be critical for periodontal tissue regeneration. In this review, we discuss the function and histological structure of the cementum to promote crystal engineering with a biochemical approach in cementum regenerative medicine. We review the microstructure of enamel and bone while discussing the mechanism underlying apatite crystal formation to infer the morphology of cementum apatite crystals and their complex structure with collagen fibers. Finally, the limitations of the current dental implant treatments in clinical practice are explored from the perspective of periodontal tissue regeneration. We anticipate the possibility of advancing periodontal tissue regenerative medicine via cementum regeneration using a combination of material science and biochemical methods.     

Application of induced pluripotent stem (iPS) cells in periodontal tissue regeneration

Tissue engineering provides a new paradigm for periodontal tissue regeneration in which proper stem cells and effective cellular factors are very important. The objective of this study was, for the first time, to investigate the capabilities and advantages of periodontal tissue regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). In this study the effect of EMD gel on iPS cells in vitro was first determined, and then tissue engineering technique was performed to repair periodontal defects in three groups: silk scaffold only; silk scaffold + EMD; and silk scaffold + EMD + iPS cells. EMD greatly enhanced the mRNA expression of Runx2 but inhibited the mRNA expression of OC and mineralization nodule formation in vitro. Transplantation of iPS cells showed higher expression levels of OC, Osx, and Runx2 genes, both 12 and 24 days postsurgery. At 24 days postsurgery in the iPS cell group, histological analysis showed much more new alveolar bone and cementum formation with regenerated periodontal ligament between them. The results showed the commitment role that EMD contributes in mesenchymal progenitors to early cells in the osteogenic lineage. iPS cells combined with EMD provide a valuable tool for periodontal tissue engineering, by promoting the formation of new cementum, alveolar bone, and normal periodontal ligament. J. Cell. Physiol. 226: 150–157, 2010. © 2010 Wiley‐Liss, Inc.     

Auto-transplanted mesenchymal stromal cell fate in periodontal tissue of beagle dogs

Background aimsMesenchymal stromal cells (MSC) possess multilineage differentiation potential and characteristics of self-renewal. It has been reported that MSC can acquire characteristics of cells in the periodontal ligament (PDL) in vitro. Moreover, the transplantation of MSC has been shown to be a promising strategy for treating periodontal defects. However, little is known about the fate of MSC in periodontal tissue in vivo. The aim of this study was to trace the paths of MSC after transplantation into periodontal tissues in vivo.MethodsMSC labeled with bromodeoxyuridine (BrdU) were transplanted into periodontal defects of beagle dogs. Six weeks after surgery, the animals were killed and decalcified specimens were prepared. Migration and differentiation of MSC were detected by single/double immunohistochemistry and a combination of immunohistochemistry and in situ hybridization.ResultsBrdU-labeled MSC were observed distributing into periodontal tissue that included alveolar bone, PDL, cementum and blood vessels and expressing surface markers typical of osteoblasts and fibroblasts.ConclusionsCumulatively, our data suggest that MSC migrate throughout periodontal tissue and differentiate into osteoblasts and fibroblasts after transplantation into periodontal defects at 6 weeks in vivo, and have the potential to regenerate periodontal tissue.     

Adipose-derived stem cells: Implications in tissue regeneration

Adipose-derived stem cells (ASCs) are mesenchymal stem cells (MSCs) that are obtained from abundant adipose tissue, adherent on plastic culture flasks, can be expanded in vitro, and have the capacity to differentiate into multiple cell lineages. Unlike bone marrow-derived MSCs, ASCs can be obtained from abundant adipose tissue by a minimally invasive procedure, which results in a high number of cells. Therefore, ASCs are promising for regenerating tissues and organs damaged by injury and diseases. This article reviews the implications of ASCs in tissue regeneration.     

Combined treatment with platelet-rich plasma and brain-derived neurotrophic factor-overexpressing bone marrow stromal cells supports axonal remyelination in a rat spinal cord hemi-section model

Background aimsCombining biologic matrices is becoming a better choice to advance stem cell-based therapies. Platelet-rich plasma (PRP) is a biologic product of concentrated platelets and has been used to promote regeneration of peripheral nerves after injury. We examined whether PRP could induce rat bone marrow stromal cells (BMSCs) differentiation in vitro and whether a combination of BMSCs, PRP and brain-derived neurotrophic factor (BDNF) could provide additive therapeutic benefits in vivo after spinal cord injury (SCI).MethodsBMSCs and BDNF-secreting BMSCs (BDNF-BMSCs) were cultured with PRP for 7 days and 21 days, respectively, and neurofilament (NF)-200, glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2) and ribosomal protein S6 kinase (p70S6K) gene levels were assessed. After T10 hemi-section in 102 rats, 15-μL scaffolds (PRP alone, BMSCs, PRP/BMSCs, BDNF-BMSCs or PRP/BDNF-BMSCs) were transplanted into the lesion area, and real-time polymerase chain reaction, Western blot, immunohistochemistry and ultrastructural studies were performed.ResultsThe messenger RNA expression of NF-200, GFAP, MAP2 and p70S6K was promoted in BMSCs and BDNF-BMSCs after culture with PRP in vitro. BDNF levels were significantly higher in the injured spinal cord after implantation of BDNF-BMSCs. In the PRP/BDNF-BMSCs group at 8 weeks postoperatively, more GFAP was observed, with less accumulation of astrocytes at the graft-host interface. Rats that received PRP and BDNF-BMSC implants showed enhanced hind limb locomotor performance at 8 weeks postoperatively compared with control animals, with more axonal remyelination.ConclusionsA combined treatment comprising PRP and BDNF-overexpressing BMSCs produced beneficial effects in rats with regard to functional recovery after SCI through enhancing migration of astrocytes into the transplants and axonal remyelination.     

Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells

It is known that the dental follicle (DF) consists of progenitor cells that give rise to the cementum, periodontal ligament, and alveolar bone; but little information is available about the regulation of DF cell differentiation into either cementogenic or osteogenic cell lineages for the regeneration of diseased periodontal tissue. Here, we investigated the roles of DF, Hertwig’s epithelial root sheath (HERS), and pulp cells in the cementum and during alveolar bone formation. We cultured these cells; transplanted them alone or in combination into immunocompromised mice; and observed their effects at 6 and 12 weeks. Histological and immunohistochemical results revealed that DF cells formed cementum-like tissues with immunoreactivity to cementum-derived attached protein, bone sialoprotein, type I collagen, and alkaline phosphatase. In addition, HERS cells played a role in the induction and maturation of cementum-like tissues formed by DF cells. In contrast, implants of DF cells in the presence of pulp cells led to the formation of bone-like tissues. Interestingly, in the presence of both HERS and pulp cells, DF cells formed both cementum-like and bone-like tissues. We demonstrated that while HERS cells are able to induce DF cell differentiation into cementoblasts and promote cementum formation, pulp cells could direct DF cell differentiation into osteoblasts and enhance alveolar bone formation. These results suggest that the combined use of DF, HERS, and pulp cells could direct DF cell differentiation into cementoblasts and/or osteoblasts in vivo, thus providing a novel strategy for the successful repair and regeneration of diseased periodontal tissue.     

Stromal cell‐derived factor‐1/Exendin‐4 cotherapy facilitates the proliferation,migration and osteogenic differentiation of human periodontal ligament stem cells in vitro and promotes periodontal bone regeneration in vivo

ObjectivesStromal cell‐derived factor‐1 (SDF‐1) actively directs endogenous cell homing. Exendin‐4 (EX‐4) promotes stem cell osteogenic differentiation. Studies revealed that EX‐4 strengthened SDF‐1‐mediated stem cell migration. However, the effects of SDF‐1 and EX‐4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo.MethodsCell‐counting kit‐8 (CCK8), transwell assay, qRT‐PCR and western blot were used to determine the effects and mechanism of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF‐1 and systemic injection of EX‐4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo.ResultsSDF‐1/EX‐4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis‐related gene expression compared to SDF‐1 or EX‐4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF‐1/EX‐4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF‐1/EX‐4 cotherapy significantly promoted new bone formation, recruited more CXCR4⁺ cells and CD90⁺/CD34^‐ stromal cells to the defects, enhanced early‐stage osteoclastogenesis and osteogenesis‐related markers expression in regenerated bone compared to control, SDF‐1 or EX‐4 in vivo.ConclusionsSDF‐1/EX‐4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration.     

Human Ng2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid‐lys scaffold fabricate a skeletal muscle tissue

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross‐linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2⁺ ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2^? ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT‐PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut‐4, and PPAR‐γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2⁺ ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre‐differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration. J. Cell. Physiol. 228: 1762–1773, 2013. © 2013 Wiley Periodicals, Inc.