Expression analysis of genes involved in the expansion of hematopoietic stem cells (SCF, Flt3-L, TPO, IL-3, and IL-6) in unrestricted somatic stem cells cultured on fibrin.

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB-HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.

In vitro release of silver ions and expression of osteogenic genes by MC3T3-E1 cell line cultured on nano-hydroxyapatite and silver/strontium-coated titanium plates.

Attempts are ongoing to improve the surface properties of dental implants by application of different coatings, aiming to enhance osseointegration, and decrease the adverse effects of titanium and its alloys used in dental implants. Coating of implant surface with hydroxyapatite (HA) is one suggested strategy for this purpose due to its high biocompatibility and similar structure to the adjacent bone. This study aimed to quantify the release of silver ions and expression of osteogenic genes by MC3T3-E1 cells cultured on nano-HA and silver/strontium (Ag/Sr)-coated titanium plates via the electrochemical deposition method. Plates measuring 10 × 10 × 0.9 mm were fabricated from Ti-6Al-4 V alloy, and polished with silicon carbide abrasive papers before electrochemical deposition to create a smooth, mirror-like surface. After applying homogenous nano-HA coatings with/without silver/strontium on the surface of the plates, the composition of coatings was confirmed by energy-dispersive X-ray spectroscopy (EDS), and their morphological properties were analyzed by scanning electron microscopy (SEM). The coated specimens were then immersed in simulated body fluid (SBF), and the concentration of released sliver ions was quantified by spectroscopy at 7-14 days. The MC3T3-E1 osteoblastic cell line was cultured in osteogenic medium for 7-14 days, and after RNA extraction and cDNA synthesis, the expression of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN); osteogenic genes was quantified by polymerase chain reaction (PCR) using SYBR Green Master Mix kit. The expression of genes and the released amount of silver ions were compared between the two groups using the Mann-Whitney U test. The two groups were not significantly different regarding silver ion release at 14 days (P > 0.05). However, silver ion release was significantly higher from nano-HA coatings with silver/strontium at 7 days (P = 0.03). The difference in expression of RUNX2 (P = 0.04), OPN (P = 0.04), and OCN (P = 0.03) genes was also significant between nano-HA coating groups with and without silver/strontium at 7 days, and the expressions were higher in nano-HA with silver/strontium group, but this difference was not significant at 14 days. Addition of silver and strontium to specimens coated with nano-HA increased the release of silver ions within the non-toxic range, and enhanced the expression of osteogenic genes particularly after 7 days.

Growth Factors in Periodontal Complex Regeneration.

The ultimate goal of periodontal treatments is the regeneration of all lost periodontal tissues including bone, cementum and the periodontal ligament (PDL). Until now, the clinical methods for periodontal regeneration have been associated with significant failure or incomplete success. Various studies have reported the promising effects of growth factors/cytokines on periodontal regeneration. Growth factors/cytokines include proteins or steroid hormones that bind to cellular receptors, known as signalling molecules, and that trigger cellular responses that eventually stimulate cell proliferation and differentiation. The present review aims to provide an overview of the main growth factors that play an important role in and have been used in the regeneration of periodontal components.

Prefabrication technique by preserving a muscular pedicle from masseter muscle as an in vivo bioreactor for reconstruction of mandibular critical-sized bone defects in canine models.

In vivo bioreactors serve as regenerative niches that improve vascularization and regeneration of bone grafts. This study has evaluated the masseter muscle as a natural bioreactor for ßTCP or PCL/ßTCP scaffolds, in terms of bone regeneration. The effect of pedicle preservation, along with sole, or MSC- or rhBMP2-combined application of scaffolds, has also been studied. Twenty-four mongrel dogs were randomly placed in six groups, including ßTCP, ßTCP/rhBMP2, ßTCP/MSCs, PCL/ßTCP, PCL/ßTCP/rhBMP2, and PCL/ßTCP/MSCs. During the first surgery, the scaffolds were implanted into the masseter muscle for being prefabricated. After 2 months, each group was divided into two subgroups prior to mandibular bone defect reconstruction; one with a preserved vascularized pedicle and one without. After 12 weeks, animals were euthanized, and new bone formation was evaluated using histological analysis. Histological analysis showed that all ß-TCP scaffold groups had resulted in significantly greater rates of new bone formation, either with a pedicle surgical approach or non-pedicle surgical approach, comparing to their parallel groups of ßTCP/PCL scaffolds (p ≤ .05). Pedicled ß-TCP scaffold groups that were treated with either rhBMP2 (48.443% ± 0.250%) or MSCs (46.577% ± 0.601%) demonstrated the highest rates of new bone formation (p ≤ .05). Therefore, masseter muscle can be used as a local in vivo bioreactor with potential clinical advantages in reconstruction of human mandibular defects. In addition, scaffold composition, pedicle preservation, and treatment with MSCs or rhBMP2, influence new bone formation and scaffold degradation rates in the prefabrication technique.

Dental stem cell banking: Techniques and protocols.

Dental tissue-derived stem cells (DSCs) provide an easy, accessible, relatively noninvasive promising source of adult stem cells (ASCs), which brought encouraging prospective for their clinical applications. DSCs provide a perfect opportunity to apply for a patient's own ASC, which poses a low risk of immune rejection. However, problems associated with the long-term culture of stem cells, including loss of proliferation and differentiation capacities, senescence, genetic instability, and the possibility of microbial contamination, make cell banking necessary. With the rapid development of advanced cryopreservation technology, various international DSC banks have been established for both research and clinical applications around the world. However, few studies have been published that provide step-by-step guidance on DSCs isolation and banking methods. The purpose of this review is to present protocols and technical details for all steps of cryopreserved DSCs, from donor selection, isolation, cryopreservation, to characterization and quality control. Here, the emphasis is on presenting practical principles in accordance with the available valid guidelines.

Comparison of osteogenic differentiation potential of induced pluripotent stem cells and buccal fat pad stem cells on 3D-printed HA/ß-TCP collagen-coated scaffolds.

Production of a 3D bone construct with high-yield differentiated cells using an appropriate cell source provides a reliable strategy for different purposes such as therapeutic screening of the drugs. Although adult stem cells can be a good source, their application is limited due to invasive procedure of their isolation and low yield of differentiation. Patient-specific human-induced pluripotent stem cells (hiPSCs) can be an alternative due to their long-term self-renewal capacity and pluripotency after several passages, resolving the requirement of a large number of progenitor cells. In this study, a new biphasic 3D-printed collagen-coated HA/ß-TCP scaffold was fabricated to provide a 3D environment for the cells. The fabricated scaffolds were characterized by the 3D laser scanning digital microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and mechanical test. Then, the osteogenesis potential of the hiPSC-seeded scaffolds was investigated compared to the buccal fat pad stem cell (BFPSC)-seeded scaffolds through in vitro and in vivo studies. In vitro results demonstrated up-regulated expressions of osteogenesis-related genes of RUNX2, ALP, BMP2, and COL1 compared to the BFPSC-seeded scaffolds. In vivo results on calvarial defects in the rats confirmed a higher bone formation in the hiPSC-seeded scaffolds compared to the BFPSC-seeded groups. The immunofluorescence assay also showed higher expression levels of collagen I and osteocalcin proteins in the hiPSC-seeded scaffolds. It can be concluded that using the hiPSC-seeded scaffolds can lead to a high yield of osteogenesis, and the hiPSCs can be used as a superior stem cell source compared to BFPSCs for bone-like construct bioengineering.

Cartilage regeneration with dual-drug-releasing injectable hydrogel/microparticle system: In vitro and in vivo study.

In this study, we developed an injectable in situ forming hydrogel/microparticle system consisting of two drugs, melatonin and methylprednisolone, to investigate the capability of the system for chondrogenesis in vitro and in vivo. The chemical, mechanical, and rheological properties of the hydrogel/microparticle were investigated. For in vitro evaluation, the adipose-derived stem cells might be mixed with hydrogel/microparticles, then cellular viability was analyzed by acridine orange/propidium iodide and 4',6-diamidino-2-phenylindole staining and also dimethylmethylene blue assay were conducted to find the amount of proteoglycan. The real-time polymerase chain reaction for aggrecan, sex-determining region Y-Box 9, collagen I (COL1), and COL2 gene expression was performed after 14 and 21 days. For evaluation of cartilage regeneration, the samples were implanted in rabbit knees with cartilaginous experimental defects. Defects were created in both knees of three groups of rabbits. Group 1 was the control with no injection, and Groups 2 and 3 were loaded with hydrogel/cell and hydrogel/microparticle/cell; respectively. Then, after 3 and 6 months, histological evaluations of the defected sites were carried out. The amount of glycosaminoglycans after 14 and 21 days increased significantly in hydrogels/microparticles loaded with cells. The expression of marker genes was also significant in hydrogels/microparticles loaded with cells. According to histology analysis, the hydrogels/microparticles loaded with cells showed the best cartilage regeneration. Overall, our study revealed that the developed injectable hydrogel/microparticle can be used for cartilage regeneration.

Fabrication of Decellularized Engineered Extracellular Matrix through Bioreactor-Based Environment for Bone Tissue Engineering.

Extracellular matrix (ECM)-contained grafts can be achieved by decellularization of native bones or synthetic scaffolds. Limitations associated with harvesting the native bone has raised interest in preparing in vitro ECM bioscaffold for bone tissue engineering. Here, we intend to develop an ECM-contained construct via decellularizing an engineered gelatin-coated ß-tricalcium phosphate (gTCP) scaffold. In order to find an optimal protocol for decellularization of cell-loaded gTCP scaffolds, they were seeded with buccal fat pad-derived stem cells. Then, four decellularization protocols including sodium dodecyl sulfate, trypsin, Triton X-100, and combined solution methods were compared for the amounts of residual cells and remnant collagen and alteration of scaffold structure. Then, the efficacy of the selected protocol in removing cells from gTCP scaffolds incubated in a rotating and perfusion bioreactor for 24 days was evaluated and compared with static condition using histological analysis. Finally, decellularized scaffolds, reloaded with cells, and their cytotoxicity and osteoinductive capability were evaluated. Complete removal of cells from gTCP scaffolds was achieved from all protocols. However, treatment with Triton X-100 showed significantly higher amount of remnant ECM. Bioreactor-incubated scaffolds possessed greater magnitude of ECM proteins including collagen and glycosaminoglycans. Reseeding the decellularized scaffolds also represented higher osteoinductivity of bioreactor-based scaffolds. Application of Triton X-100 as decellularization protocol and usage of bioreactors are suggested as a suitable technique for designing ECM-contained grafts for bone tissue engineering.

Computational modeling of media flow through perfusion-based bioreactors for bone tissue engineering.

Bioreactor system has been used in bone tissue engineering in order to simulate dynamic nature of bone tissue environments. Perfusion bioreactors have been reported as the most efficient types of shear-loading bioreactor. Also, combination of forces, such as rotation plus perfusion, has been reported to enhance cell growth and osteogenic differentiation. Mathematical modeling using sophisticated infrastructure processes could be helpful and streamline the development of functional grafts by estimating and defining an effective range of bioreactor settings for better augmentation of tissue engineering. This study is aimed to conduct computational modeling for newly designed bioreactors in order to alleviate the time and material consuming for evaluating bioreactor parameters and effect of fluid flow hydrodynamics (various amounts of shear stress) on osteogenesis. Also, biological assessments were performed in order to validate similar parameters under implementing the perfusion or rotating and perfusion fluid motions in bioreactors' prototype. Finite element method was used to investigate the effect of hydrodynamic of fluid flow inside the bioreactors. The equations used in the simulation to calculate the velocity values and consequently the shear stress values include Navier-Stokes and Brinkman equations. It has been shown that rotational fluid motion in rotating and perfusion bioreactor produces more velocity and shear stress compared with perfusion bioreactor. Moreover, implementing the perfusion together with rotational force in rotating and perfusion bioreactors has been shown to have more cell proliferation and higher activity of alkaline phosphatase enzyme as well as formation of extra cellular matrix sheet, as an indicator of bone-like tissue formation.

Egg shell-derived calcium phosphate/carbon dot nanofibrous scaffolds for bone tissue engineering: Fabrication and characterization.

Recent exciting findings of the particular properties of Carbon dot (CDs) have shed light on potential biomedical applications of CDs-containing composites. While CDs so far have been widely used as biosensors and bioimaging agents, in the present study for the first time, we evaluate the osteoconductivity of CDs in poly (ε-caprolactone) (PCL)/polyvinyl alcohol (PVA) [PCL/PVA] nanofibrous scaffolds. Moreover, further studies were performed to evaluate egg shell-derived calcium phosphate (TCP3) and its cellular responses, biocompatibility and in vitro osteogenesis. Scaffolds were fabricated by simultaneous electrospinning of PCL with three different types of calcium phosphate, PVA and CDs. Fabricated scaffolds were characterized by Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), contact angle measurement and degradation assessment. SEM, the methyl thiazolyl tetrazolium (MTT) assay, and alkaline phosphatase (ALP) activity test were performed to evaluate cell morphology, proliferation and osteogenic differentiation, respectively. The results demonstrated that while the addition of just 1 wt% CDs and TCP3 individually into PCL/PVA nanocomposite enhanced ALP activity and cell proliferation rate (p < 0.05), the synergetic effect of CDs/TCP3 led to highest osteogenic differentiation and proliferation rate compared to other scaffolds (p < 0.05). Hence, CDs and PCL/PVA-TCP3 could serve as a potential candidate for bone tissue regeneration.

Buccal fat pad-derived stem cells with anorganic bovine bone mineral scaffold for augmentation of atrophic posterior mandible: An exploratory prospective clinical study.

BACKGROUND: Application of adipose-derived stem cells originated from buccal fat pad (BFP) can simplify surgical procedures and diminish clinical risks compared to large autograft harvesting. PURPOSE: This study sought to evaluate and compare the efficacy of buccal fat pad-derived stem cells (BFPSCs) in combination with anorganic bovine bone mineral (ABBM) for vertical and horizontal augmentation of atrophic posterior mandibles. MATERIALS AND METHODS: Fourteen patients with atrophic posterior mandible were elected for this prospective exploratory study. BFP (3-5 mL) was harvested and BFPSCs were isolated and combined with ABBM at 50% ratio. The vertical and horizontal alveolar deficiencies were augmented by 50% mixture of ABBM with either BFPSCs (group 1) or particulated autologous bone (group 2). Titanium mesh was contoured to the desired 3D shape of the alveolar ridge and fixated to the host sites over the graft material of the two groups. At first, the amount of new bone areas was calculated by quantitative analysis of cone beam computed tomography (CBCT) images that were taken 6 months postoperatively according to regenerative techniques (group 1 vs group 2 without considering the type of bone defects). Second, these amounts were calculated in each group based on the type of defects. RESULTS: Quantitative analysis of CBCT images revealed the areas of new bone formation were 169.5 ± 5.90 mm2 and 166.75 ± 10.05 mm2 in groups 1 and 2, respectively. The area of new bone formation for vertical defects were 164.91 ± 3.74 mm2 and 169.36 ± 12.09 mm2 in groups 1 and 2, respectively. The area of new bone formation for horizontal deficiencies were 170.51 ± 4.54 mm2 and 166.98 ± 9.36 mm2 in groups 1 and 2, respectively. There were no statistically significant differences between the two groups in any of the pair-wise comparisons (P > 0.05). CONCLUSIONS: The findings of the present study demonstrated lack of difference in bone volume formation between BFPSCs and autologous particulate bone in combination with ABBM. If confirmed by future large-scale clinical trial, BFPSCs may provide an alternative to autogenous bone for reconstruction of alveolar ridge defects.

In vitro effect of graphene structures as an osteoinductive factor in bone tissue engineering: A systematic review.

Graphene and its derivatives have been well-known as influential factors in differentiating stem/progenitor cells toward the osteoblastic lineage. However, there have been many controversies in the literature regarding the parameters effect on bone regeneration, including graphene concentration, size, type, dimension, hydrophilicity, functionalization, and composition. This study attempts to produce a comprehensive review regarding the given parameters and their effects on stimulating cell behaviors such as proliferation, viability, attachment and osteogenic differentiation. In this study, a systematic search of MEDLINE database was conducted for in vitro studies on the use of graphene and its derivatives for bone tissue engineering from January 2000 to February 2018, organized according to the PRISMA statement. According to reviewed articles, different graphene derivative, including graphene, graphene oxide (GO) and reduced graphene oxide (RGO) with mass ratio ≤1.5 wt % for all and concentration up to 50 µg/mL for graphene and GO, and 60 µg/mL for RGO, are considered to be safe for most cell types. However, these concentrations highly depend on the types of cells. It was discovered that graphene with lateral size less than 5 µm, along with GO and RGO with lateral dimension less than 1 µm decrease cell viability. In addition, the three-dimensional structure of graphene can promote cell-cell interaction, migration and proliferation. When graphene and its derivatives are incorporated with metals, polymers, and minerals, they frequently show promoted mechanical properties and bioactivity. Last, graphene and its derivatives have been found to increase the surface roughness and porosity, which can highly enhance cell adhesion and differentiation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2284-2343, 2018.

Buccal Fat Pad as a Potential Source of Stem Cells for Bone Regeneration: A Literature Review.

Adipose tissues hold great promise in bone tissue engineering since they are available in large quantities as a waste material. The buccal fat pad (BFP) is a specialized adipose tissue that is easy to harvest and contains a rich blood supply, and its harvesting causes low complications for patients. This review focuses on the characteristics and osteogenic capability of stem cells derived from BFP as a valuable cell source for bone tissue engineering. An electronic search was performed on all in vitro and in vivo studies that used stem cells from BFP for the purpose of bone tissue engineering from 2010 until 2016. This review was organized according to the PRISMA statement. Adipose-derived stem cells derived from BFP (BFPSCs) were compared with adipose tissues from other parts of the body (AdSCs). Moreover, the osteogenic capability of dedifferentiated fat cells (DFAT) derived from BFP (BFP-DFAT) has been reported in comparison with BFPSCs. BFP is an easily accessible source of stem cells that can be obtained via the oral cavity without injury to the external body surface. Comparing BFPSCs with AdSCs indicated similar cell yield, morphology, and multilineage differentiation. However, BFPSCs proliferate faster and are more prone to producing colonies than AdSCs.

Application of selected scaffolds for bone tissue engineering: a systematic review.

PURPOSE: The current systematic review investigated the results of application of some of the most commonly used scaffolds in conjugation with stem cells and growth factors in animal and clinical studies. METHODS: A comprehensive electronic search was conducted according to the PRISMA guidelines in NCBI PMC and PubMed from January 1970 to December 2015 limited to English language publications with available full texts. In vivo studies in relation to "bone healing," "bone regeneration," and at least one of the following items were investigated: allograft, ß-tricalcium phosphate, deproteinized bovine bone mineral, hydroxyapetite/tricalcium phosphate, nanohydroxyapatite, and composite scaffolds. RESULTS: A total of 1252 articles were reviewed, and 46 articles completely fulfilled the inclusion criteria of this study. The highest bone regeneration has been achieved when combination of all three elements, given scaffolds, mesenchymal stem cells, and growth factors, were used. Among studies being reported in this review, bone marrow mesenchymal stem cells are the most studied mesenchymal stem cells, ß-tricalcium phosphate is the most frequently used scaffold, and platelet-rich plasma is the most commonly used growth factor. CONCLUSION: The current review aimed to inform reconstructive surgeons of how combinations of various mesenchymal stem cells, scaffolds, and growth factors enhance bone regeneration. The highest bone regeneration has been achieved when combination of all three elements, given scaffolds, mesenchymal stem cells, and growth factors, were used.

Impact of Tissue Harvesting Sites on the Cellular Behaviors of Adipose-Derived Stem Cells: Implication for Bone Tissue Engineering.

The advantages of adipose-derived stem cells (AdSCs) over bone marrow stem cells (BMSCs), such as being available as a medical waste and less discomfort during harvest, have made them a good alternative instead of BMSCs in tissue engineering. AdSCs from buccal fat pad (BFP), as an easily harvestable and accessible source, have gained interest to be used for bone regeneration in the maxillofacial region. Due to scarcity of data regarding comparative analysis of isolated AdSCs from different parts of the body, we aimed to quantitatively compare the proliferation and osteogenic capabilities of AdSCs from different harvesting sites. In this study, AdSCs were isolated from BFP (BFPdSCs), abdomen (abdomen-derived mesenchymal stem cells (AbdSCs)), and hip (hip-derived mesenchymal stem cells (HdSCs)) from one individual and were compared for surface marker expression, morphology, growth rate, and osteogenic differentiation capability. Among them, BFPdSCs demonstrated the highest proliferation rate with the shortest doubling time and also expressed vascular endothelial markers including CD34 and CD146. Moreover, the expression of osteogenic markers were significantly higher in BFPdSCs. The results of this study suggested that BFPdSCs as an encouraging source of mesenchymal stem cells are to be used for bone tissue engineering.

Fabrication of a three-dimensional ß-tricalcium-phosphate/gelatin containing chitosan-based nanoparticles for sustained release of bone morphogenetic protein-2: Implication for bone tissue engineering.

Fabrication of an ideal scaffold having proper composition, physical structure and able to have sustained release of growth factors still is challenging for bone tissue engineering. Current study aimed to design an appropriate three-dimensional (3-D) scaffold with suitable physical characteristics, including proper compressive strength, degradation rate, porosity, and able to sustained release of bone morphogenetic protein-2 (BMP2), for bone tissue engineering. A highly porous 3-D ß-tricalcium phosphate (ß-TCP) scaffolds, inside of which two perpendicular canals were created, was fabricated using foam-casting technique. Then, scaffolds were coated with gelatin layer. Next, BMP2-loaded chitosan (CS) nanoparticles were dispersed into collagen hydrogel and filled into the scaffold canals. Physical characteristics of fabricated constructs were evaluated. Moreover, the capability of given construct for bone regeneration has been evaluated in vitro in interaction with human buccal fat pad-derived stem cells (hBFPSCs). The results showed that gelatin-coated TCP scaffold with rhBMP2 delivery system not only could act as a mechanically and biologically compatible framework, but also act as an osteoinductive graft by sustained delivering of rhBMP2 in a therapeutic window for differentiation of hBFPSCs towards the osteoblast lineage. The proposed scaffold model can be suggested for delivering of cells and other growth factors such as vascular endothelial growth factor (VEGF), alone or in combination, for future investigations.

Induced pluripotent stem cells as a new getaway for bone tissue engineering: A systematic review.

OBJECTIVES: Mesenchymal stem cells (MSCs) are frequently used for bone regeneration, however, they are limited in quantity. Moreover, their proliferation and differentiation capabilities reduce during cell culture expansion. Potential application of induced pluripotent stem cells (iPSCs) has been reported as a promising alternative source for bone regeneration. This study aimed to systematically review the available literature on osteogenic potential of iPSCs and to discuss methods applied to enhance their osteogenic potential. METHODS AND MATERIALS: A thorough search of MEDLINE database was performed from January 2006 to September 2016, limited to English-language articles. All in vitro and in vivo studies on application of iPSCs in bone regeneration were included. RESULTS: The current review is organized according to the PRISMA statement. Studies were categorized according to three different approaches used for osteo-induction of iPSCs. Data are summarized and reported according to the following variables: types of study, cell sources used for iPSC generation, applied reprogramming methods, applied osteo-induction methods and treatment groups. CONCLUSION: According to the articles reviewed, osteo-induced iPSCs revealed osteogenic capability equal to or superior than MSCs; cell sources do not significantly affect osteogenic potential of iPSCs; addition of resveratrol to the osteogenic medium (OM) and irradiatiation after osteogenic induction reduce teratoma formation in animal models; transfection with lentiviral bone morphogenetic protein 2 results in higher mineralization compared to osteo-induction in OM; addition of TGF-ß, IGF-1 and FGF-ß to OM increases osteogenic capability of iPSCs.

Evaluation of bone regeneration potential of dental follicle stem cells for treatment of craniofacial defects.

BACKGROUND AIMS: Stem cell-based tissue regeneration offers potential for treatment of craniofacial bone defects. The dental follicle, a loose connective tissue surrounding the unerupted tooth, has been shown to contain progenitor/stem cells. Dental follicle stem cells (DFSCs) have strong osteogenesis capability, which makes them suitable for repairing skeletal defects. The objective of this study was to evaluate bone regeneration capability of DFSCs loaded into polycaprolactone (PCL) scaffold for treatment of craniofacial defects. METHODS: DFSCs were isolated from the first mandibular molars of postnatal Sprague-Dawley rats and seeded into the PCL scaffold. Cell attachment and cell viability on the scaffold were examined with the use of scanning electron microscopy and alamar blue reduction assay. For in vivo transplantation, critical-size defects were created on the skulls of 5-month-old immunocompetent rats, and the cell-scaffold constructs were transplanted into the defects. RESULTS: Skulls were collected at 4 and 8 weeks after transplantation, and bone regeneration in the defects was evaluated with the use of micro-computed tomography and histological analysis. Scanning electron microscopy and Alamar blue assay demonstrated attachment and proliferation of DFSCs in the PCL scaffold. Bone regeneration was observed in the defects treated with DFSC transplantation but not in the controls without DFSC transplant. Transplanting DFSC-PCL with or without osteogenic induction before transplantation achieved approximately 50% bone regeneration at 8 weeks. Formation of woven bone was observed in the DFSC-PCL treatment group. Similar results were seen when osteogenic-induced DFSC-PCL was transplanted to the critical-size defects. CONCLUSIONS: This study demonstrated that transplantation of DFSCs seeded into PCL scaffolds can be used to repair craniofacial defects.

The role of dentin matrix protein 1 (DMP1) in regulation of osteogenic differentiation of rat dental follicle stem cells (DFSCs).

OBJECTIVES: Primary isolated dental follicle stem cells (DFSCs) possess a strong osteogenesis capability, and such capability is reduced during in vitro culture. Because dentin matrix protein 1 (DMP1) is essential in the maturation of osteoblasts, our objectives were to determine (1) the expression of DMP1 in the DFSCs, (2) the correlation between DMP1 expression and osteogenic capability of DFSCs, and (3) the ability of DMP1 to promote osteogenic differentiation of DFSCs. METHODS: DFSCs and their non-stem cell counterpart dental follicle cells (DFC) were established from postnatal rat pups. Expression of DMP1 in the DFSCs and DFC was determined using real-time RT-PCR and western blotting. Different passages of DFSCs were subjected to osteogenic induction. The correlation between osteogenesis and DMP1 expression was analyzed. Then, expression of DMP1 in the DFSCs was knocked-down using siRNA, followed by osteogenic induction to evaluate the effect of DMP1-knockdown. Finally, the late passage DFSCs with reduced DMP1 expression and osteogenic capability were cultured in osteogenic induction medium containing mouse recombinant DMP1 (mrDMP1) to determine if DMP1 can restore osteogenesis of DFSCs. RESULTS: DFSCs expressed much higher levels of DMP1 than did DFC. DMP1 expression was correlated with the osteogenic capability of DFSCs. Knockdown of DMP1 expression markedly decreased the osteogenesis and osteogenic gene expression in the DFSCs whereas adding mrDMP1 protein to the osteogenic induction medium enhanced osteogenesis. CONCLUSIONS: DMP1 is highly expressed in the DFSCs, but minimally expressed in non-stem cell DFC. DMP1 appears to play an important role for osteogenic differentiation of the DFSCs.