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.

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.

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.

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.

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.