Mouse Calvarial Defect Research Articles

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model.

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.

Biofabrication and application of decellularized bone extracellular matrix for effective bone regeneration

In order to promote effective healing of a bone defect, various bone grafts have been widely implanted into defect sites. However, several drawbacks, such as immunologic rejection, insufficient tissue healing, or secondary damage to the donor site, are still concerns. These limitations of the current bone grafts can be overcome by use of a natural ECM that can influence basic cellular behaviors, such as proliferation, migration, and differentiation. Herein, we introduce a novel bone decellularized ECM (bdECM) material extracted from bovine bones via demineralization and decellularization. This sequential process enabled us to prepare raw material possessing higher contents of native BMP-2 and BMP-7, and a fibrous microarchitecture resembling the collagen bundle in the ECM was maintained. Furthermore, residual calcium and phosphate originated from the bovine bone were considerably removed, and contamination by residual DNA was significantly reduced during the fabrication process. These features enabled the scaffold successfully promote mineralization of the cultured primary osteoblasts in vitro, which was significantly better than those from the bone powder or demineralized bone matrix. In a mouse calvarial defect, new bone formation was improved in the bdECM group and the density of those was also higher than other groups.

The promotion of bone regeneration through CS/GP-CTH/antagomir-133a/b sustained release system

Few studies reported the application of miRNA in bone regeneration. In this study, the expression of miR133a and miR133b in murine BMSCs was inhibited via antagomiR-133a/b and the osteogenic differentiation in murine BMSCs was evaluated. The RT-PCR, flow cytometry, cell counting kit-8, and annexin V-FITC/PI double staining assays were performed. Double knockdown miR133a and miR133b can promote BMSC osteogenic differentiation. At optimum N/P ration (15:1), the loading efficiency can reach over 90%. CTH-antagomiR-133a/b showed no cytotoxicity to BMSCs and diminished miR133a and miR133b expression in BMSCs. Furthermore, chitosan-based sustained delivery system can facilitate continuous dosing of antagomiR-133a/b, which enhanced calcium deposition and osteogenic specific gene expression in vitro. The new bone formation was enhanced after the sustained delivery system containing CTH-antagomiR-133a/b nanoparticles was used in mouse calvarial bone defect model. Our results demonstrate that CTH nanoparticles could facilitate continuous dosing of antagomiR133a/b, which can promote osteogenic differentiation.

In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells.

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties.

55: Bone Regeneration Assessment Through SPECT/CT Imaging in a Mouse Calvarial Defect Model

Introduction The evaluation of novel synthetic bone implants relies mostly on histological examination, but as bone repair takes several weeks, non-invasive imaging techniques have gained interest in the recent past. Ιn the frame of this study, we establish a SPECT/CT imaging protocol for the longitudinal assessment of bone healing of a calvarial mouse defect model using mineralized collagen-based scaffolds. Materials and Methods Two symmetrical calvarial bone defects, 1.5 mm in diameter each, were created in female Swiss mice. Gaps were monitored weekly through SPECT/CT imaging (γ-CUBE, X-CUBE, Molecubes, providing a resolution of 0.6 mm and 0.05 mm, respectively), using [99mTc]-MDP for 5 weeks. Defects created on the left part of the mouse skull were left unfilled (control group), whereas those on the right part were filled with mineralized horse collagen scaffold materials (HA/Coll). Results and Discussion: The monitoring of bone healing was successfully and accurately performed via SPECT/CT imaging. CT imaging showed the actual bone filling process and the formation of new bone tissue, whereas SPECT imaging provided the increased metabolic activity. For the HA/Coll treated defect, a peak in metabolic activity was noted at week 1 post-surgery and the defect was filled by newly developed bone tissue by week 3 post-surgery (Figure 1). In contrast, the metabolic activity at the control defect area was stable but significantly lower and a complete healing was not observed even up to week 5post-surgery. These results show that combined SPECT/CT imaging can provide an accurate tool for bone healing monitoring. CT is a high resolution tool for new bone formation control and SPECT is a high sensitivity tool for early metabolic activity confirmation, also having a high prognostic value for bone healing. Conclusions SPECT revealed the earliest signs of new bone tissue formation due to the increased metabolic activity and CT provided a clear visualization and quantification ability of the healing process over time. This study is part of the EU Horizon 2020 research and innovation VIVOIMAG project under the Marie Skłodowska-Curie grant agreement No 645757. This study was co-supported through the Programme of Industrial Scholarships of Stavros Niarchos Foundation. Figure 1. Indicative axial CT (a), sagittal SPECT/CT (b) and axial SPECT/CT (c) at 3 h [99m]Tc-MDP post-injection (~2 mCi) of a bilateral calvarial mouse defect treated (HA/Coll) and non-treated (control), at week 1 (first row) and at week 3 (second row) post-surgery.

Clumps of Mesenchymal Stem Cell/Extracellular Matrix Complexes Generated with Xeno-Free Conditions Facilitate Bone Regeneration via Direct and Indirect Osteogenesis.

Three-dimensional clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. We demonstrated previously that C-MSCs can be transplanted into bone defect regions with no artificial scaffold to induce bone regeneration. To apply C-MSCs in a clinical setting as a reliable bone regenerative therapy, the present study aimed to generate C-MSCs in xeno-free/serum-free conditions that can exert successful bone regenerative properties and to monitor interactions between grafted cells and host cells during bone healing processes. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. Then, C-MSCs were transplanted into an immunodeficient mouse calvarial defect model. Transplantation of C-MSCs induced bone regeneration in a time-dependent manner. Immunofluorescence staining showed that both donor human cells and host mice cells contributed to bone reconstruction. Decellularized C-MSCs implantation failed to induce bone regeneration, even though the host mice cells can infiltrate into the defect area. These findings suggested that C-MSCs generated in xeno-free/serum-free conditions can induce bone regeneration via direct and indirect osteogenesis.

Microporous methacrylated glycol chitosan-montmorillonite nanocomposite hydrogel for bone tissue engineering

Injectable hydrogels can fill irregular defects and promote in situ tissue regrowth and regeneration. The ability of directing stem cell differentiation in a three-dimensional microenvironment for bone regeneration remains a challenge. In this study, we successfully nanoengineer an interconnected microporous networked photocrosslinkable chitosan in situ-forming hydrogel by introducing two-dimensional nanoclay particles with intercalation chemistry. The presence of the nanosilicates increases the Young’s modulus and stalls the degradation rate of the resulting hydrogels. We demonstrate that the reinforced hydrogels promote the proliferation as well as the attachment and induced the differentiation of encapsulated mesenchymal stem cells in vitro. Furthermore, we explore the effects of nanoengineered hydrogels in vivo with the critical-sized mouse calvarial defect model. Our results confirm that chitosan-montmorillonite hydrogels are able to recruit native cells and promote calvarial healing without delivery of additional therapeutic agents or stem cells, indicating their tissue engineering potential.

Stimulatory Effects of KPR-A148 on Osteoblast Differentiation and Bone Regeneration.

Xanthine derivatives have been used to treat a variety of medical conditions including respiratory disease and neural degeneration. However, few studies have reported their effects on bone regeneration. Therefore, we investigated the effects of KPR-A148, a synthetic xanthine derivative on osteoblast differentiation in vitro and bone regeneration in vivo. The cytotoxicity of KPR-A148 was evaluated using MC3T3-E1 cells by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltertrazolium bromide assay. The effects of KPR-A148 on osteoblast differentiation were examined by alkaline phosphatase staining, Alizarin red S staining, and real-time PCR of osteoblast differentiation marker genes. To investigate the effects of KPR-A148 on in vivo bone regeneration, a KPR-A148-containing collagen sponge was implanted into a mouse calvarial defect and KPR-A148 was injected twice, weekly. Bone regeneration was evaluated quantitatively by micro-CT and qualitatively by hematoxylin and eosin, as well as Masson's Trichrome staining. KPR-A148 did not show toxicity in the MC3T3-E1 cells and promoted osteoblast differentiation in a concentration-dependent manner. 10μM of KPR-A148 showed the most significant effect on alkaline phospatase staining and matrix mineralization. KPR-A148 increased the expression of osteoblast marker genes in both the early and late stages of differentiation. In addition, KPR-A148 significantly induced new bone formation in the calvarial defect model. These results demonstrate that KPR-A148 strongly induces osteoblast differentiation and new bone formation. Therefore, it could be used as a potential therapeutic agent for regenerating bone following its destruction by disease or trauma.

Bioinspired and osteopromotive polydopamine nanoparticle-incorporated fibrous membranes for robust bone regeneration

Due to their inherent hydrophobic and bioinert nature, synthetic degradable polymer-based membranes show inferior stem cell attachment, proliferation, and even differentiation. To overcome these limitations, bioinspired and osteopromotive polydopamine nanoparticle-incorporated fibrous membranes are developed via a two-step route: pH-induced polymerization of dopamine and co-electrospinning of polycaprolactone (PCL) with polydopamine nanoparticles (PDA NPs). Hybrid membranes with optimized PDA NP content exhibit high quantities of apatite deposition and prominent cytocompatibility (cell attachment, spreading and reproduction) and osteo-differentiation potential (alkaline phosphatase activity, calcium mineralization, and osteogenesis-related genes and protein expression) of human mesenchymal stem cells cultured without any growth factors. Importantly, in vivo assessments using a mouse calvarial critical-sized defect demonstrate that the engineered fibrous membranes remarkably boost bone reconstruction and regeneration. Accordingly, our bioinspired PCL-based hybrid fibrous membranes with robust osteoinductive ability can potentially be utilized as a clinically applicable candidate in guided tissue regeneration applications.

Identification of cell-penetrating osteogenic peptide from copine-7 protein and its delivery system for enhanced bone formation.

Peptide and proteins are recognized as highly selective and therapeutically active biomaterials, as well as relatively safe in clinical application. A calcium phospholipid-binding protein, copine 7 (CPNE7), has been recently identified to induce hard tissue regeneration, including bone and dentin by internalizing into the cells. However, the clinical application of the full length of CPNE7 has limited due to its large size with short half-life. Herein, as an alternative to CPNE7, six bioactive synthetic peptides are designed from CPNE7 (CPNE7-derived peptides, CDP1-CDP6) and investigated their osteogenic potential. Among the CDPs, CDP4 have the highest level of cell-penetrating activity as well as osteogenic efficiency in dental pulp stem cells (DPSCs). CDP4 increased the expression of osteogenesis-related genes and proteins, which was comparable to that by BMP-2. The cell penetration capacity of CDP4 may synergistically induce the osteogenic potential of DPSCs. Moreover, the implantation of the mixture of CDP4 with injectable collagen gel increased bone formation with recovery in the mouse calvarial defect model, comparable to full-length CPNE7 and even BMP-2. In conclusion, these results suggest that our synthetic peptide, CDP4, can be applied in combination with biomaterial to provide high osteogenic efficacy in the field of bone tissue engineering.

In vitro bone-like nodules generated from patient-derived iPSCs recapitulate pathological bone phenotypes.

The recapitulation of bone formation via the in vitro generation of bone-like nodules is frequently used to understand bone development. However, current bone-induction techniques are slow and difficult to reproduce. Here, we report the formation of bone-like nodules within ten days, via the use of retinoic acid (RA) to induce the osteogenic differentiation of human induced pluripotent stem cells (hiPSCs) into osteoblast-like and osteocyte-like cells that create human bone tissue when implanted in calvarial defects in mice. We also show that the induction of bone formation depends on cell signalling through the RA receptors RARα and RARβ, which simultaneously activate the BMP (bone morphogenetic protein) and Wnt signalling pathways. Moreover, by using patient-derived hiPSCs, the bone-like nodules recapitulated the osteogenesis-imperfecta phenotype, which was rescued via the correction of disease-causing mutations and partially by an mTOR (mechanistic target of rapamycin) inhibitor. The method of inducing bone nodules may serve as a fast and reproducible model for the study of the formation of both healthy and pathological bone.

Potential therapeutic use of relaxin in accelerating closure of cranial bone defects in mice.

Bone fractures are associated with considerable morbidity and increased mortality. A major limitation to healing is lack of bone blood flow, which is impaired by physical disruption of intraskeletal and/or periosteal vasculature by the fracture. Thus, pharmacological interventions are needed to improve osseous blood flow, thereby accelerating bone fracture closure. Relaxin is secreted by the ovary and circulates in rodents and humans during pregnancy. Because relaxin might benefit bone fracture healing by stimulating angiogenesis, vasculogenesis (and potentially osteogenesis) through mobilization and activation of bone marrow progenitor cells, and by increasing blood flow via vasodilation, we investigated whether relaxin administration would accelerate closure of a calvarial defect in mice. Whether administered systemically by osmotic pump or locally by collagen scaffolds for ~2 week period after lesioning, relaxin did not accelerate bone healing. Despite implementing relaxin doses that reached plasma concentrations spanning the physiological to supraphysiological range, testing the closure of two different sizes of calvarial lesions, allowing for different intervals of time from instigation of cranial lesion to euthanasia, and investigating mice of different ages, we did not observe a significant benefit of relaxin in bone lesion healing. Nor did we observe stimulation of blood vessel formation in the bone lesion by the hormone. An incidental finding was that relaxin appeared to enhance trabecular bone growth in an uninjured control bone (femur). Although the results of this study were not supportive of a therapeutic benefit for relaxin on calvarial defect closure, future investigation is needed employing different animal species and experimental models of bone fracture.

Histological Criteria that Distinguish Human and Mouse Bone Formed Within a Mouse Skeletal Repair Defect.

The effectiveness of autologous cell-based skeletal repair continues to be controversial in part because in vitro predictors of in vivo human bone formation by cultured human progenitor cells are not reliable. To assist in the development of in vivo assays of human osteoprogenitor potential, a fluorescence-based histology of nondecalcified mineralized tissue is presented that provides multiple criteria to distinguish human and host osteoblasts, osteocytes, and accumulated bone matrix in a mouse calvarial defect model. These include detection of an ubiquitously expressed red fluorescent protein reporter by the implanted human cells, antibodies specific to human bone sialoprotein and a human nuclear antigen, and expression of a bone/fibroblast restricted green fluorescent protein reporter in the host tissue. Using low passage bone marrow-derived stromal cells, robust human bone matrix formation was obtained. However, a striking feature is the lack of mouse bone marrow investment and osteoclasts within the human bone matrix. This deficiency may account for the accumulation of a disorganized human bone matrix that has not undergone extensive remodeling. These features, which would not be appreciated by traditional decalcified paraffin histology, indicate the human bone matrix is not undergoing active remodeling and thus the full differentiation potential of the implanted human cells within currently used mouse models is not being realized.

Osteogenic Effects of VEGF-Overexpressed Human Adipose-Derived Stem Cells with Whitlockite Reinforced Cryogel for Bone Regeneration.

Bone is a vascularized tissue that is comprised of collagen fibers and calcium phosphate crystals such as hydroxyapatite (HAp) and whitlockite (WH). HAp and WH are known to elicit bone regeneration by stimulating osteoblast activities and osteogenic commitment of stem cells. In addition, vascular endothelial growth factor (VEGF) is shown to promote osteogenesis and angiogenesis which is considered as an essential process in bone repair by providing nutrients. In this study, VEGF-secreting human adipose-derived stem cells (VEGF-ADSCs) are developed by transducing ADSCs with VEGF-encoded lentivirus. Additionally, WH-reinforced gelatin/heparin cryogels (WH-C) are fabricated by loading WH into gelatin/heparin cryogels. VEGF-ADSC secrete tenfold more VEGF than ADSC and show increased VEGF secretion with cell growth. Also, incorporation of WH into cryogels provides a mineralized environment with ions secreted from WH. When the VEGF-ADSCs are seeded on WH-C, sustained release of VEGF is observed due to the specific affinity of VEGF to heparin. Finally, the synergistic effect of VEGF-ADSC and WH on osteogenesis is successfully confirmed by alkaline phosphatase and real-time polymerase chain reaction analysis. In vivo bone formation is demonstrated via implantation of VEGF-ADSC seeded WH-C into mouse calvarial bone defect model, resulted in enhanced bone development with the highest bone volume/total volume.

Characterization of the chondrogenic and osteogenic potential of male and female human muscle-derived stem cells: Implication for stem cell therapy.

People of all backgrounds are susceptible to bone and cartilage damage, and these injuries can be debilitating. Current treatments for bone and cartilage injuries are less than optimal, and we are interested in developing new approaches to treat these diseases, specifically using human muscle-derived stem cells (hMDSCs). Our lab previously demonstrated that sex differences exist between male and female murine MDSCs; thus, this paper sought to investigate whether sex differences also exist in hMDSCs. In the present study, we characterized the chondrogenic and osteogenic sex differences of hMDSCs in vitro and in vivo. We performed in vitro osteogenic and chondrogenic differentiation using hMDSC pellet cultures. As demonstrated by microCT, histology, and immunohistochemistry, male hMDSCs were more chondrogenic and osteogenic than their female counterparts in vitro. No differences were observed based on the sex of hMDSCs in osteogenic and chondrogenic gene expression and cell surface markers. For our in vivo study, we transduced hMDSCs with lenti-BMP2/GFP and transplanted these cells into critical-sized calvarial defects in mice. MicroCT results revealed that male hMDSCs regenerated more bone at 2 weeks and demonstrated higher bone density at 4 and 6 weeks than female hMDSCs. Histology demonstrated that both male and female hMDSCs regenerated functional bone. Clinical relevance: These studies reinforce that stem cells isolated from male and female patients differ in function, and we should disclose the sex of cells used in future studies. Considering sex differences of hMDSCs may help to improve cell-based therapies for autologous cell treatment of bone and cartilage damage. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1339-1349, 2019.

Oxidative Epigallocatechin Gallate Coating on Polymeric Substrates for Bone Tissue Regeneration.

Plant derived flavonoids have not been well explored in tissue engineering applications due to difficulties in efficient formulations with biomaterials for controlled presentation. Here, the authors report that surface coating of epigallocatechin gallate (EGCG) on polymeric substrates including poly (L-lactic acid) (PLLA) nanofibers can be performed via oxidative polymerization of EGCG in the presence of cations, enabling regulation of biological functions of multiple cell types implicated in bone regeneration. EGCG coating on the PLLA nanofiber promotes osteogenic differentiation of adipose-derived stem cells (ADSCs) and is potent to suppress adipogenesis of ADSCs while significantly reduces osteoclastic maturation of murine macrophages. Moreover, EGCG coating serves as a protective layer for ADSCs against oxidative stress caused by hydrogen peroxide. Finally, the in vivo implantation of EGCG-coated nanofibers into a mouse calvarial defect model significantly promotes the bone regeneration (61.52±28.10%) as compared to defect (17.48±11.07%). Collectively, the results suggest that EGCG coating is a simple bioinspired surface modification of polymeric biomaterials and importantly can thus serve as a promising interface for tuning activities of multiple cell types associated with bone fracture healing.

Extracellular enzymatically synthesized glycogen promotes osteogenesis by activating osteoblast differentiation via Akt/GSK-3β signaling pathway.

Glycogen is the stored form of glucose and plays a major role in energy metabolism. Recently, it has become clear that enzymatically synthesized glycogen (ESG) has biological functions, such as the macrophage-stimulating activity. This study aimed to evaluate the effect of ESG on osteogenesis. MC3T3-E1 cells were cultured with ESG, and their cell proliferative activity and osteoblast differentiation were measured. An in vivo study was conducted in which ESG pellets with BMP-2 were grafted into mouse calvarial defects and histomorphometrically analyzed for the new bone formation. To confirm the effect of ESG on bone growth in vivo, ESG was orally administered to pregnant mice and the femurs of their pups were examined. We observed that ESG stimulated cell proliferation and enhanced messenger RNA expression of osteocalcin and osteopontin in MC3T3-E1 cells. ESG was taken up by the cells associated with GLUT-1 and activated the Akt/GSK-3β pathway. In vivo, the new bone formation in the calvarial defect was significantly accelerated by ESG and the maternal administration of ESG promoted fetal bone growth. In conclusion, ESG stimulates cell proliferation and differentiation of preosteoblasts via the activation of Akt/GSK-3β signaling and promotes new bone formation in vivo, suggesting that ESG could be a useful stimulant for osteogenesis.

Bioceramic akermanite enhanced vascularization and osteogenic differentiation of human induced pluripotent stem cells in 3D scaffolds in vitro and vivo.

A growing number of studies suggest that the modulation of cell differentiation by biomaterials is critical for tissue engineering. In previous work, we demonstrated that human induced pluripotent stem cells (iPSCs) are remarkably promising seed cells for bone tissue engineering. In addition, we found that the ionic products of akermanite (Aker) are potential inducers of osteogenic differentiation of iPSCs. Furthermore, composite scaffolds containing polymer and bioceramics have more interesting properties compared to pure bioceramic scaffolds for bone tissue engineering. The characteristic of model biomaterials in bone tissue engineering is their ability to control the osteogenic differentiation of stem cells and simultaneously induce the angiogenesis of endothelia cells. Thus, this study aimed at investigating the effects of poly(lactic-co-glycolic acid)/Aker (PLGA-Aker) composite scaffolds on angiogenic and osteogenic differentiation of human iPSCs in order to optimize the scaffold compositions. The results from Alizarin Red S staining, qRT-PCR analysis of osteogenic genes (BMP2, RUNX2, ALP, COL1 and OCN) and angiogenic genes (VEGF and CD31) demonstrated that PLGA/Aker composite scaffolds containing 10% Aker exhibited the highest stimulatory effects on the osteogenic and angiogenic differentiation of human iPSCs among all scaffolds. After the scaffolds were implanted in nu/nu mice subcutaneous pockets and calvarial defects, H&E staining, BSP immunostaining, qRT-PCR analysis and micro-CT analysis (BMD, BV/TV) indicated that PLGA + 10% Aker scaffolds enhanced the vascularization and osteogenic differentiation of human iPSCs and stimulated the repair of bone defects. Taken together, our work indicated that combining scaffolds containing silicate bioceramic Aker and human iPSCs is a promising approach for the enhancement of bone regeneration.

Electrosprayed naringin-loaded microsphere/SAIB hybrid depots enhance bone formation in a mouse calvarial defect model

The burst release of active osteogenic factors, which is not beneficial to osteogenesis, is commonly encountered in bone tissue engineering. The aims of this study were to prepare naringin-loaded microsphere/sucrose acetate isobutyrate (Ng-m-SAIB) hybrid depots, reduce the burst release of naringin (Ng), and improve osteogenesis. The morphology and size distributions of electrosprayed Ng-microspheres were characterized by scanning electron microscopy (SEM). The Ng-microspheres and Ng-m-SAIB depots were characterized by Fourier transform infrared spectroscopy (FTIR) and in vitro release studies. In vitro osteoblast-microsphere interactions and in vivo osteogenesis were assessed after implantation of Ng-m-SAIB depots. The addition of sucrose acetate isobutyrate (SAIB) to monodisperse Ng-microspheres did not cause a change in the chemical structure. The performances of the microspheres in osteoblast-microsphere interactions were better when the naringin content was 4% than when it was at 2% and 6%. On the first day following the loading of Ng-microspheres (2%, 4%, and 6%) into SAIB depots, the burst release was reduced dramatically from 70.9% to 6.3%, 73.1% to 7.2%, and 73.9% to 9.9%, respectively. In addition, after 8 weeks, the new bone formation rate in the calvarial defects of SD rats receiving Ng-m-SAIB was 53.1% compared to 21.2% for the control group and 16.1% for the microsphere-SAIB group. These results demonstrated that Ng-m-SAIB hybrid depots may have promise in bone regeneration applications.

Multi-layered PLLA-nanosheets loaded with FGF-2 induce robust bone regeneration with controlled release in critical-sized mouse femoral defects

To overcome clinical issues caused by large bone defects and subsequent nonunion, various approaches to bone regeneration have been researched, including tissue engineering, biomaterials, stem cells and drug screening. Previously, we developed a free-standing biodegradable polymer nanosheet composed of poly(L-lactic acid) (PLLA) using a simple fabrication process consisting of spin-coating and peeling techniques. We reported that sandwich-type PLLA nanosheets loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) displayed long-lasting, sustained release of rhBMP-2, and markedly enhanced bone regeneration in mouse calvarial bone defects. Here, we fabricated multi-layered nanosheets loaded with fibroblast growth factor-2 (FGF-2), and investigated their application for long bone regeneration. Subcutaneously implanted tri-layered PLLA nanosheets displayed sustained release of loaded rhFGF-2 for about 2 weeks. Next, we prepared critical-sized mouse femoral defects and implanted mono- or tri-layered nanosheets, or a gelatin hydrogel with rhFGF-2. Amongst these conditions, the tri-layered nanosheet most efficiently induced bone regeneration. Indeed, bone regeneration was enhanced even after 4 weeks in the tri-layered nanosheet group, and was accompanied by FGFR1 activation and subsequent osteoblast differentiation. Multi-layered PLLA nanosheets loaded with rhFGF-2 may be useful for bone regenerative medicine. Furthermore, the multi-layered PLLA nanosheet structure may potentially be applied as a potent sustained-release carrier. Statements of SignificanceHere, we describe multi-layered poly(L-lactic acid) (PLLA) nanosheets loaded with recombinant human fibroblast growth factor-2 (rhFGF-2) as a modified sustained-release carrier for bone regeneration. In vivo imaging system analysis revealed that subcutaneously implanted tri-layered PLLA nanosheets displayed sustained release of loaded rhFGF-2 for 2 weeks. In critical-sized mouse femoral defects, tri-layered nanosheets loaded with rhFGF-2 most efficiently induced bone regeneration. Notably, bone regeneration was enhanced even after 4 weeks in the tri-layered nanosheet group, and was accompanied by FGFR1 activation and subsequent osteoblast differentiation. Multi-layered PLLA nanosheets loaded with rhFGF-2 may be useful for bone regenerative medicine. Furthermore, the multi-layered PLLA nanosheet structure may potentially be applied as a potent sustained-release carrier.