Stable gene expression by self-complementary adeno-associated viruses in human MSCs

Genetically modified mesenchymal stem cells (MSCs) are potentially valuable tools for the novel treatment of human illnesses. Here, we investigated whether gene transfers by self-complementary adeno-associated viruses (scAAV) lead to promising genetic modification in human bone marrow and umbilical cord blood MSCs. Of the various scAAVs, scAAV2, and scAAV5 effectively and safely expressed transgenes in both hMSCs. Transduction efficiency with scAAV2 at 1000 multiplicity of infection was 66.3+/-9.4% and 67.6+/-6.7% in bone marrow and umbilical cord blood MSCs, respectively. A co-infection study showed that the distinct scAAV2 and scAAV5 can effectively express different transgenes in the same hMSC. hMSCs transduced by scAAVs showed long-term gene expression for three months in rat brains. Genetic modification by scAAVs did not affect osteogenic differentiation of hMSCs. Therefore, the present study strongly supports the promising potential of scAAVs as a technical platform for safe, long-term transgene expression in hMSCs.     

Enhanced osteoinduction by mesenchymal stem cells transfected with a fiber-mutant adenoviral BMP2 gene

BACKGROUND: Bone regeneration therapy using mesenchymal stem cells (MSCs) is beginning to come into clinical use. To overcome the difficulty of healing large bone defects, we previously reported the efficacy of using rat mesenchymal stem cells (rMSCs) carrying a modified adenoviral vector (Adv-F/RGD) with an RGD-containing peptide in the HI loop of the fiber knob domain of adenovirus type 5 (Ad5). METHODS: Firstly, we evaluated the transduction efficiency of Adv-F/RGD into bone-marrow-derived human MSCs (hMSCs) using a beta-galactosidase chemiluminescent assay. Next, we evaluated whether the vector AxCAhBMP2-F/RGD carrying the human bone morphogenetic protein 2 (BMP2) gene could enhance the osteogenic activity of hMSCs in vitro and in vivo (in an ectopic model). In the ectopic model, transduced hMSCs, hMSCs in the presence of recombinant human BMP2 (rhBMP2) or hMSCs alone were implanted into a subcutaneous site of nude mice. We also applied this vector system to an orthotopic model (large bone defect model) using rMSCs. RESULTS: The transduction efficiency of Adv-F/RGD into hMSCs was increased 10-fold over the vector containing the wild-type fiber (Adv-F/wt), as assessed by a beta-galactosidase chemiluminescent assay. AxCAhBMP2-F/RGD increased the osteogenic activity of hMSCs in vitro. In the ectopic model, AxCAhBMP2-F/RGD-transduced hMSCs were found to induce new bone at 1 week after transplantation, and a greater quantity of new bone was formed than in other groups. Similarly, AxCAhBMP2-F/RGD-transduced rMSCs induced a greater quantity of new bone than other groups (AxCAhBMP2-F/wt-transduced rMSCs, rMSCs in the presence of rhBMP2, rMSCs alone, or scaffolds alone) in the orthotopic model. CONCLUSIONS: These data suggest that Adv-F/RGD is useful for introducing foreign genes into MSCs and that it will be a powerful gene therapy tool for bone regeneration and other tissue-engineering applications.     

Promotion of human mesenchymal stem cell differentiation on bioresorbable polycaprolactone/biphasic calcium phosphate composite scaffolds for bone tissue engineering

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ～ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.     

Osteogenic response to BMP-2 of hMSCs grown on apatite-coated scaffolds

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to non-mineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration.     

Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?

Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.     

Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.     

Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these results suggest that microporous electrospun scaffolds pre-seeded with fibroblasts promote greater wound-healing than acellular scaffolds.     

BMP2 cross-linked by transglutaminase 2 to collagen-plla scaffold promotes osteogenic differentiation in mesenchymal stem cells

Transglutaminase 2 (TG2) was used to attach biologically-active BMP2 to collagen type I-coated poly-l-lactic acid (PLLA) nanofibrous scaffolds. Irreversibly cross-linked BMP2 retained its activity and induced Smad-dependent gene expression in cells seeded on PLLA–BMP2 scaffolds. These modified scaffolds promote osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) cultured in low-serum and growth factor free medium and support deposition of the calcified matrix and induction of the molecular osteogenic markers Runx2, osteopontin, osteonectin and bone sialoprotein. Importantly, the PLLA–BMP2 scaffolds did not support chondrogenic differentiation in hBMSCs as there was no expression of chondrogenic markers aggrecan, Sox 9, and collagen type II, and no deposition of cartilaginous glycosaminoglycan-rich matrix. Thus, TG2-mediated cross-linking of BMP2 to a scaffold is a novel approach to induce osteoblast-specific programming of hBMSCs in a spatially controlled manner.     

Delivery of platelet-derived growth factor as a chemotactic factor for mesenchymal stem cells by bone-mimetic electrospun scaffolds

The recruitment of mesenchymal stem cells (MSCs) is a vital step in the bone healing process, and hence the functionalization of osteogenic biomaterials with chemotactic factors constitutes an important effort in the tissue engineering field. Previously we determined that bone-mimetic electrospun scaffolds composed of polycaprolactone, collagen I and nanohydroxyapatite (PCL/col/HA) supported greater MSC adhesion, proliferation and activation of integrin-related signaling cascades than scaffolds composed of PCL or collagen I alone. In the current study we investigated the capacity of bone-mimetic scaffolds to serve as carriers for delivery of an MSC chemotactic factor. In initial studies, we compared MSC chemotaxis toward a variety of molecules including PDGF-AB, PDGF-BB, BMP2, and a mixture of the chemokines SDF-1α, CXCL16, MIP-1α, MIP-1β, and RANTES. Transwell migration assays indicated that, of these factors, PDGF-BB was the most effective in stimulating MSC migration. We next evaluated the capacity of PCL/col/HA scaffolds, compared with PCL scaffolds, to adsorb and release PDGF-BB. We found that significantly more PDGF- BB was adsorbed to, and subsequently released from, PCL/col/HA scaffolds, with sustained release extending over an 8-week interval. The PDGF-BB released was chemotactically active in transwell migration assays, indicating that bioactivity was not diminished by adsorption to the biomaterial. Complementing these studies, we developed a new type of migration assay in which the PDGF-BB-coated bone-mimetic substrates were placed 1.5 cm away from the cell migration front. These experiments confirmed the ability of PDGF-BB-coated PCL/col/HA scaffolds to induce significant MSC chemotaxis under more stringent conditions than standard types of migration assays. Our collective results substantiate the efficacy of PDGF-BB in stimulating MSC recruitment, and further show that the incorporation of native bone molecules, collagen I and nanoHA, into electrospun scaffolds not only enhances MSC adhesion and proliferation, but also increases the amount of PDGF-BB that can be delivered from scaffolds.     

miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have self-renewal and differentiation capabilities but the regulatory mechanisms of MSC fate determination remain poorly understood. Here, we aimed to identify microRNAs enriched in hMSCs that modulate differentiation commitments. Microarray analysis revealed that miR-140-5p is commonly enriched in undifferentiated hMSCs from various tissue sources. Moreover, bioinformatic analysis and luciferase reporter assay validated that miR-140-5p directly represses bone morphogenic protein 2 (BMP2). Furthermore, blocking miR-140-5p in hMSCs increased the expression of BMP signaling components and critical regulators of osteogenic differentiation. We propose that miR-140-5p functionally inhibits osteogenic lineage commitment in undifferentiated hMSCs.     

A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col,PLLA/HA,and PLLA/Col for Tissue Engineering Application

A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200–950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.     

Donepezil hydrochloride as a novel inducer for osteogenic differentiation of mesenchymal stem cells on PLLA scaffolds in vitro

Over the past decades, bone defects caused by illness or trauma have been the most common traumatic injuries in humans and treatment of orthopedic infections has always been a serious challenge to experts in the world. In this project, poly L-lactic acid (PLLA) nanofibrous scaffolds were synthesized as a nontoxic, eco-friendly, and cost-effective scaffold by the electrospinning technique. Then, the impact of PLLA on the cell proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assayed in the presence and absence of donepezil hydrochloride (DH) which was prescribed in patients with Alzheimer's disease. Also, hMSCs were seeded on PLLA scaffold in the presence (PLLA-DH) and absence of 1 μg mL^-1 of DH under osteogenic induction media. Osteogenic differentiation of hMSCs was assessed by specific bone-related tests including alkaline phosphatase (ALP) activity, Alizarin red and von Kossa staining, calcium content assay. Also, Osteocalcin and osteopontin were evaluated as osteogenic proteins as well as ALP, osteonectin, osteocalcin, collagen type I (Col-I) and Runx2 as osteogenic genes via immunocytochemistry (ICC) and Real-time PCR analyses. The obtained data showed the higher ALP enzyme activity and biomineralization, more intensity during von Kossa staining as well as the increase in the expression rate of osteogenic related gene and protein markers in differentiated hMSCs on PLLA-DH. In conclusion, the present study revealed that the combination of PLLA scaffold with DH provides a scope to develop a suitable matrix in bone tissue engineering applications.     

Bone morphogenetic protein activity preservation with extracorporeal irradiation- and liquid nitrogen freezing-treated recycled autografts for biological reconstruction in malignant bone tumor

Recycled autografts have been commonly used in biological reconstruction in conjunction with wide bone resection. Extracorporeal irradiation (ECIR) and freezing are the two major options for pretreating tumor-bearing autografts before transplant. This study, for the first time, compared the effects of these two techniques on bone morphogenetic protein (BMP)-2 activity. Bone tissue extracted from human femoral heads were treated through either ECIR at different doses (5000, 15,000, and 30,000 rad) or liquid nitrogen (LN) freezing for different durations (5, 10, and 15 min). The amount of BMP was analyzed through enzyme-linked immunosorbent assay (ELISA assay). Furthermore, we also used tandem mass spectrometry to analyze change of BMP-2 and BMP-7 expression after high dosage of irradiation (30,000 rad) and long-time of freezing (15 min). To directly evaluate the effect of ECIR or LN freezing treatment on the activity of BMP, commercial recombinant human BMP-2 (rhBMP-2) was added to the culture of human mesenchymal stem cells (hMSCs). The post-treatment activity of rhBMP-2 was quantitated by measuring the osteogenic differentiation of hMSCs with Alizarin Red S staining. Through Western blotting, the activation of the BMP signaling pathway by phospho-Smad antibodies was analyzed. Our results showed that post-treatment levels of BMP did not differ among the ECIR and LN freezing treatments in ELISA assay, but tandem mass spectrometry showed significantly lower expression of BMP-2 after 30,000 rad of irradiation. Both ECIR and freezing lowered the expression of regulatory factors involved in the BMP-activated signaling cascades and similar results were also observed in osteogenic differentiation of hMSCs. However, LN freezing preserved better bioactivity of rhBMP-2 whereas dosage-dependent declination was observed in ECIR groups. In conclusion, considering BMP-2 activity, LN freezing-treated autografts may result in a better osteoinduction outcomes than those treated using ECIR. Further investigation of the factors involved in bone formation is required.     

The response of human mesenchymal stem cells to osteogenic signals and its impact on bone tissue engineering

Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.     

The promising effects of BMP2 transfected mesenchymal stem cells on human osteosarcoma

Selective targeting of transfected mesenchymal stem cells (MSCs) carrying specific antioncogenes to the tumor was suggested as a treatment option. Bone morphogenetic protein-2 (BMP2) was shown to inhibit the proliferation and aggressiveness of osteosarcoma (OS) cells. Here, we aimed to assess the homing efficiency of intraperitoneally administered hMSCs transfected with BMP2 to the tumoral site and their effects on OS using an orthotopic xenograft murine model. Orthotopic xenograft murine model of OS in six-week-old female NOD/SCID mice using 143B cells was established. hMSCs transfected with BMP2 (BMP2⁺hMSC) were used. In vivo experiments performed on four groups of mice that received no treatment, or intraperitoneally administered BMP2, hMSCs, and BMP2⁺hMSCs. Histopathological and immunohistochemical studies were used to evaluate the pathological identification and to assess the dimensions and necrotic foci of the tumor, the features of lung metastases, and immunostaining against p27, Ki-67, and caspase-3 antibodies. The osteogenic differentiation markers BMP2, BMP4, COL1A1, OPN, OCN and PF4 evaluated using RT-PCR. The tumor dimensions in the hMSCs group were significantly higher than those of the remaining groups (p < 0.01). The number of metastatic foci in the BMP2⁺hMSCs group was significantly lower than those of the other groups (p < 0.01). The current results showed that the intraperitoneal route could be efficiently used for targeting hMSCs to the tumoral tissues for effective BMP2 delivery. In this study, the effects of BMP2 transfected hMSCs on human OS and metastasis were promising for achieving osteogenic differentiation and reduced metastatic process.     

The in vivo assessment of a novel scaffold containing heparan sulfate for tissue engineering with human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and mature tissue macrophages, respectively. It was found that the survival of the implanted human cells was affected by the host response to the implant regardless of the presence of HS, highlighting the importance of controlling the host response to tissue engineering devices.     

Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.