Rat Cranial Defect Research Articles

Investigation of Vasculogenesis Inducing Biphasic Scaffolds for Bone Tissue Engineering.

Vascularization is the main obstacle for the bone tissue engineering strategies since the defect size is generally large. Incorporation of angiogenic factors is one of the strategies employed in order to accelerate vascularization and improve bone healing. In this study, a biphasic scaffold consisting of fibrous poly(lactide-co-glycolide) (PLGA) and poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) hydrogel loaded with vascular endothelial growth factor-A (VEGF) inducer, GS4012, was constructed. Mesenchymal stem cells isolated from rat bone marrow (rBMSCs) were used for differentiation into osteogenic cells, and endothelial cells isolated from rat peripheral blood (rPBECs) were used to test the in vitro endothelial cell recruitment. The biphasic scaffold was tested for cell proliferation, ALP expression, VEGF induction, expression of osteogenic genes by rBMSCs, and recruitment of rPBECs in vitro and for improved bone healing and vascularization in vivo on critical size rat cranial defects. Endothelial migration through porous insert and VEGF induction were obtained in vitro in response to GS4012 as well as the upregulation of ALP, Runx2, Col I, and OC gene expressions. The biphasic scaffold was also shown to be effective in improving endothelial cell recruitment, vascularization, and bone healing in vivo. Thus, the proposed design has a great potential for the healing of critical size bone defect in tissue engineering studies according to both in vitro and in vivo investigations.

Construction of nanofibrous scaffolds with interconnected perfusable microchannel networks for engineering of vascularized bone tissue

Vascularization and bone regeneration are two closely related processes during bone reconstruction. A three-dimensional (3D) scaffold with porous architecture provides a suitable microenvironment for vascular growth and bone formation. Here, we present a simple and general strategy to construct a nanofibrous poly(l-lactide)/poly(ε-caprolactone) (PLLA/PCL) scaffold with interconnected perfusable microchannel networks (IPMs) based on 3D printing technology by combining the phase separation and sacrificial template methods. The regular and customizable microchannel patterns within the scaffolds (spacings: 0.4 mm, 0.5 mm, and 0.6 mm; diameters: 0.8 mm, 1 mm, and 1.2 mm) were made to investigate the effect of microchannel structure on angiogenesis and osteogenesis. The results of subcutaneous embedding experiment showed that 0.5/0.8-IPMs (spacing/diameter = 0.5/0.8) and 0.5/1-IPMs (spacing/diameter = 0.5/1) scaffolds exhibited more vascular network formation as compared with other counterparts. After loading with vascular endothelial growth factor (VEGF), VEGF@IPMs-0.5/0.8 scaffold prompted better human umbilical vein endothelial cells (HUVECs) migration and neo-blood vessel formation, as determined by Transwell migration, scratch wound healing, and chorioallantoic membrane (CAM) assays. Furthermore, the microangiography and rat cranial bone defects experiments demonstrated that VEGF@IPMs-0.5/0.8 scaffold exhibited better performance in vascular network formation and new bone formation compared to VEGF@IPMs-0.5/1 scaffold. In summary, our results suggested that the microchannel structure within the scaffolds could be tailored by an adjustable caramel-based template strategy, and the combination of interconnected perfusion microchannel networks and angiogenic factors could significantly enhance vascularization and bone regeneration.

Curcumin reduces apoptosis and promotes osteogenesis of human periodontal ligament stem cells under oxidative stress in vitro and in vivo

AimsHuman periodontal ligament stem cells (hPDLSCs) tether the teeth to the surrounding bone and are considered as major functional stem cells responsible for regeneration of the alveolar bone and periodontal ligament tissue. However, the outcome of stem cell regenerative therapy is affected by the survival rate and their differentiation potential of transplanted cells. This is primarily because of local oxidative stress and chronic inflammation at the transplantation site. Therefore, our study aimed to explore whether a natural antioxidant, curcumin could increase the tissue regeneration ability of transplanted hPDLSCs. Main methodsA hydrogen peroxide environment and a rat cranial bone defect model were built to mimic the oxidative stress conditions in vitro and in vivo, respectively. We evaluated the effect of curcumin on oxidative status, apoptosis, mitochondrial function and osteogenic differentiation of H2O2-stimulated hPDLSCs in vitro. We also measured the effect of curcumin on cell viability and bone repair ability of transplanted hPDLSCs in vivo. Key findingsOur data showed that curcumin enhanced cell proliferation, reduced the reactive oxygen species (ROS) levels and apoptosis, maintained the standard mitochondrial structure and function, and promoted osteogenic differentiation of H2O2-stimulated hPDLSCs. The extracellular regulated protein kinases 1/2 (Erk1/2) signaling pathway was determined to be involved in the osteogenic differentiation of the H2O2-stimulated hPDLSCs. Moreover, curcumin enhanced the viability and the bone repair ability of hPDLSCs in vivo. SignificanceCurcumin reduced apoptosis and promoted osteogenesis of the hPDLSCs under oxidative stress, and might therefore have a potential clinical use with respect to tissue regeneration.

BMP-6 carrying metal organic framework-embedded in bioresorbable electrospun fibers for enhanced bone regeneration

Biomolecule carrier structures have attracted substantial interest owing to their potential utilizations in the field of bone tissue engineering. In this study, MOF-embedded electrospun fiber scaffold for the controlled release of BMP-6 was developed for the first time, to enrich bone regeneration efficacy. The scaffolds were achieved by first, one-pot rapid crystallization of BMP-6 encapsulated ZIF-8 nanocrystals-as a novel carrier for growth factor molecules- and then electrospinning of the blending solution composed of poly (ε-caprolactone) and BMP-6 encapsulated ZIF-8 nanocrystals. BMP-6 molecule encapsulation efficiency for ZIF-8 nanocrystals was calculated as 98%. The in-vitro studies showed that, the bioactivity of BMP-6 was preserved and the release lasted up to 30 days. The release kinetics fitted the Korsmeyer-Peppas model exhibiting a pseudo-Fickian behavior. The in-vitro osteogenesis studies revealed the superior effect of sustained release of BMP-6 towards osteogenic differentiation of MC3T3-E1 pre-osteoblasts. In-vivo studies also revealed that the sustained slow release of BMP-6 was responsible for the generation of well-mineralized, new bone formation in a rat cranial defect. Our results proved that; MOF-carriers embedded in electrospun scaffolds can be used as an effective platform for bone regeneration in bone tissue engineering applications. The proposed approach can easily be adapted for various growth factor molecules for different tissue engineering applications.

Simultaneous incorporation of PTH(1–34) and nano-hydroxyapatite into Chitosan/Alginate Hydrogels for efficient bone regeneration

Tissue regeneration based on the utilization of artificial soft materials is considered a promising treatment for bone-related diseases. Here, we report cranial bone regeneration promoted by hydrogels that contain parathyroid hormone (PTH) peptide PTH(1–34) and nano-hydroxyapatite (nHAP). A combination of the positively charged natural polymer chitosan (CS) and negatively charged sodium alginate led to the formation of hydrogels with porous structures, as shown by scanning electron microscopy. Rheological characterizations revealed that the mechanical properties of the hydrogels were almost maintained upon the addition of nHAP and PTH(1–34). In vitro experiments showed that the hydrogel containing nHAP and PTH(1–34) exhibited strong biocompatibility and facilitated osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) via the Notch signaling pathway, as shown by the upregulated expression of osteogenic-related proteins. We found that increasing the content of PTH(1–34) in the hydrogels resulted in enhanced osteogenic differentiation of BMSCs. Implantation of the complex hydrogel into a rat cranial defect model led to efficient bone regeneration compared to the rats treated with the hydrogel alone or with nHAP, indicating the simultaneous therapeutic effect of nHAP and PTH during the treatment process. Both the in vitro and in vivo results demonstrated that simultaneously incorporating nHAP and PTH into hydrogels shows promise for bone regeneration, suggesting a new strategy for tissue engineering and regeneration in the future.

Fabricating a novel HLC-hBMP2 fusion protein for the treatment of bone defects

Treating serious bone trauma with an osteo-inductive agent such as bone morphogenetic proteins (BMPs) has been considered as an optimized option when delivered via a collagen sponge (CS). Previous works have shown that the BMP concentration and release rate from approved CS carriers is difficult to control with precision. Here we presented the fabrication of a recombinant fusion protein from recombinant human-like collagen (HLC) and human BMP-2 (hBMP2). The fusion protein preserved the characteristic of HLC allowing the recombinant protein to be expressed in Yeast (such as Pichia pastoris GS115) and purified rapidly and easily with mass production after methanol induction. It also kept the stable properties of HLC and hBMP2 in the body fluid environment with good biocompatibility and no cytotoxicity. Moreover, the recombinant fusion protein fabricated a vertical through-hole structure with improved mechanical properties, and thus facilitated migration of bone marrow mesenchymal stem cells (MSCs) into the fusion materials. Furthermore, the fusion protein degraded and released hBMP-2 in vivo allowing osteoinductive activity and the enhancement of utilization rate and the precise control of the hBMP2 release. This fusion protein when applied to cranial defects in rats was osteoinductively active and improved bone repairing enhancing the repairing rate 3.5- fold and 4.2- fold when compared to the HLC alone and the control, respectively. There were no visible inflammatory reactions, infections or extrusions around the implantation sites observed. Our data strongly suggests that this novel recombinant fusion protein could be more beneficial in the treatment of bone defects than the simple superposition of the hBMP2/collagen sponge.

BMSC-Exosomes Carry Mutant HIF-1α for Improving Angiogenesis and Osteogenesis in Critical-Sized Calvarial Defects.

Repair and reconstruction of critical-sized bone defects has always been a difficult task in orthopedics. Hypoxia inducible factor-1α (HIF-1α) plays an important role in bone defect repair, it has the dual function of promoting osteogenesis and vascular regeneration, but it is quickly degraded by the body under normoxic conditions. Previously we prepared mutant HIF-1α, which has been shown to efficiently maintain cellular expression under normoxic conditions. In this study, we evaluated for the first time the role of exosomes of rat bone marrow mesenchymal stem cell carry mutant HIF-1α (BMSC-Exos-HIF1α) in repairing critical-sized bone defects. Evaluation of the effects of BMSC-Exos-HIF1α on bone marrow mesenchymal stem cells (BMSCs) proliferation and osteogenic differentiation by cell proliferation assay, alkaline phosphatase activity assay, alizarin red staining, real-time quantitative polymerase chain reaction. BMSC-Exos-HIF1α was loaded onto the β-TCP stent implanted in the bone defect area using a rat cranial critical-sized bone defect model, and new bone formation and neovascularization were detected in vivo by micro-CT, fluorescence labeling analysis, Microfil perfusion, histology and immunohistochemical analysis. In vitro results showed that BMSC-Exos-HIF1α stimulated the proliferation of BMSCs and up-regulated the expression level of bone-related genes, which was superior to bone marrow MSC exosomes (BMSC-Exos). In vivo results showed that BMSC-Exos-HIF1α combined with β-TCP scaffold promoted new bone regeneration and neovascularization in the bone defect area, and the effect was better than that of BMSC-Exos combined with β-TCP scaffold. In this study, the results showed that BMSC-Exos-HIF1α stimulated the proliferation and osteogenic differentiation of BMSCs and that BMSC-Exos-HIF1α combined with β-TCP scaffolds could repair critical-sized bone defects by promoting new bone regeneration and neovascularization.

Rabbit umbilical cord mesenchymal stem cells: A new option for tissue engineering.

The source and availability of cells for tissue engineering in large scale research or clinical trials requires special attention. We propose the idea of applying rabbit umbilical cord mesenchymal stem cells for this purpose. Here, the structure of the rabbit umbilical cord was analyzed and compared to that of human umbilical cord, both macroscopically and histologically. Next, we isolated, cultured and identified the proliferative activity and immunological characteristics of rabbit umbilical cord mesenchymal stem cells in vitro using mixed lymphocyte reaction, flow cytometry and an enzyme-linked immunosorbent assay. Furthermore, we evaluated the effects of biphasic calcium phosphate ceramic scaffolds seeded with rabbit umbilical cord mesenchymal stem cells in rat cranial defect models using multiple techniques, including radiological, histological and immunohistochemistry. In vitro studies demonstated a high level of proliferation and multi-lineage differentiation potential in rabbit umbilical cord mesenchymal stem cells. Rabbit umbilical cord mesenchymal stem cells exibited low immunogenicity properties and immune suppression capability with respect to both the allogeneic and xenogeneic immune response. The results of the in vivo study showed that rabbit umbilical cord mesenchymal stem cells could promote osteogenesis in heterogeneous hosts. The rabbit umbilical cord mesenchymal stem cells may be a new source for tissue engineering.

The "Yin and Yang" of Immunomodulatory Magnesium-Enriched Graphene Oxide Nanoscrolls Decorated Biomimetic Scaffolds in Promoting Bone Regeneration.

Tissue regeneration driven by immunomodulatory agents has emerged as a potential solution for repairing bone defects. However, the therapeutic benefits are compromised by disturbances in the pro- and anti-inflammatory balance. Here, using magnesium nanoparticles (MgNPs) as a template, magnesium-enriched graphene oxide nanoscrolls (MgNPs@GNSs) designed for combinational modulation of the inflammatory response are reported. First, the different effects of graphene oxide (GO) and magnesium ions (Mg2+ ) on Raw264.7 macrophage phenotype transformation are screened. The results reveal that GO activates inflammatory M1 macrophages, and that Mg2+ facilitates repolarization of M1 macrophages to the pro-healing M2 phenotype. With sustained release of Mg2+ , the MgNPs@GNS nanoplatform can orchestrate harmonious type 1 and type 2 inflammatory responses. Mg2+ decrease the internalization of GO and downregulate the nuclear factor kappa-B pathway, which is profoundly involved in the inflammatory process. A series of experiments show that the ordered inflammatory response induced by MgNPs@GNSs stimulates in vitro angiogenesis and osteogenesis through chemotactic, mitogenic, and morphogenic actions. Obvious vascularized bone regeneration is achieved in a rat cranial bone defect model via MgNPs@GNS deposited decellularized bone matrix scaffold. Therefore, the potential of using inherently therapeutic nanomedicine to modulate biomaterial-induced immune responses and thus enhance bone regeneration is demonstrated.

Gene transfection achieved by utilizing antibacterial calcium phosphate nanoparticles for enhanced regenerative therapy

Protamine-coated multi-shell calcium phosphate (CaP) was developed as a non-viral vector for tissue regeneration therapy. CaP nanoparticles loaded with different amounts of plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) were used to treat MC3T3E1 cells, and the yield of the released BMP-2 or IGF-1 was measured using ELISA 3 days later. Collagen scaffolds containing CaP nanoparticles were implanted into rat cranial bone defects, and BMP-2 and IGF-1 yields, bone formation, and bone mineral density enhancement were evaluated 28 days after gene transfer. The antibacterial effects of CaP nanoparticles against Streptococcus mutans and Aggregatibacter actinomycetemcomitans increased with an increase in the protamine dose, while they were lower for Staphylococcus aureus and Porphyromonas gingivalis. In the combination treatment with BMP-2 and IGF-1, the concentration ratio of BMP-2 and IGF-1 is an important factor affecting bone formation activity. The calcification activity and OCN mRNA of MC3T3E1 cells subjected to a BMP-2:IGF-1 concentration ratio of 1:4 was higher at 14 days. During gene transfection treatment, BMP-2 and IGF-1 were released simultaneously after gene transfer; the loaded dose of the plasmid DNA encoding IGF-1 did not impact the BMP-2 or IGF-1 yield or new bone formation ratio in vitro and in vivo. In conclusion, two growth factor-releasing systems were developed using an antibacterial gene transfer vector, and the relationship between the loaded plasmid DNA dose and resultant growth factor yield was determined in vitro and in vivo.

Hierarchical dual-porous hydroxyapatite doped dendritic mesoporous silica nanoparticles based scaffolds promote osteogenesis in vitro and in vivo

Biomaterial based scaffolds for treating large bone defects require excellent biocompatibility and osteoconductivity. Here we report on the fabrication of hydroxyapatite-dendritic mesoporous silica nanoparticles (HA-DMSN) based scaffolds with hierarchical micro-pores (5 µm) and nano-pores (6.4 nm), and their application for bone regeneration. The in vitro studies demonstrated good biocompatibility of dissolution extracts, as well as enhanced osteogenic potential indicated by dose-dependent upregulation of bone marker gene expression (osteocalcin gene (OCN), osteopontin gene (OPN), collagen type I alpha 1 gene (CoL1A1), runt-related transcription factor 2 gene (RUNX2), and integrin-binding sialoprotein gene (IBSP)), alkaline phosphatise (ALP) activity, and alizarin red staining. The in vivo studies showed that HA-DMSN scaffolds significantly increased bone formation in a rat cranial bone defect model after 4 weeks healing. Our study provides a simple method to fabricate promising inorganic scaffolds with hierarchical pores for bone tissue engineering.

Hybrid gelatin/oxidized chondroitin sulfate hydrogels incorporating bioactive glass nanoparticles with enhanced mechanical properties, mineralization, and osteogenic differentiation

Biopolymer based hydrogels are characteristic of their biocompatibility and capability of mimicking extracellular matrix structure to support cellular behavior. However, these hydrogels suffer from low mechanical properties, uncontrolled degradation, and insufficient osteogenic activity, which limits their applications in bone regeneration. In this study, we developed hybrid gelatin (Gel)/oxidized chondroitin sulfate (OCS) hydrogels that incorporated mesoporous bioactive glass nanoparticles (MBGNs) as bioactive fillers for bone regeneration. Gel-OCS hydrogels could be self-crosslinked in situ under physiological conditions in the presence of borax. The incorporation of MBGNs enhanced the crosslinking and accelerated the gelation. The gelation time decreased with increasing the concentration of MBGNs added. Incorporation of MBGNs in the hydrogels significantly improved the mechanical properties in terms of enhanced storage modulus and compressive strength. The injectability of the hydrogels was not significantly affected by the MBGN incorporation. Also, the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro and rat cranial defect restoration in vivo were significantly promoted by the hydrogels in the presence of MBGNs. The hybrid Gel-OCS/MBGN hydrogels show promising potential as injectable biomaterials or scaffolds for bone regeneration/repair applications given their tunable degradation and gelation behavior as well as favorable mechanical behavior and osteogenic activities.

Development of electrospun polymer scaffolds for the localized and controlled delivery of siponimod for the management of critical bone defects.

Sphingosine 1-phosphate (S1P) receptor modulators can influence bone regeneration owing to their positive impact on osteoblast differentiation and neovascularisation. While previous studies have utilised non-specific S1P and fingolimod, this study aims to design and characterise a controlled release vehicle to deliver the specific S1P1 & 5 receptor modulator siponimod and test its effectiveness in rat critical cranial defects. Electrospun scaffolds of poly lactide-co-glycolide (PLGA) were loaded with siponimod at drug:polymer mass ratios of 0.5:100 to 2:100. Where indicated, collagen was co-spun at a collagen:polymer mass ratio of 2:100. Thereafter, scaffolds underwent in vitro physicochemical characterisation and in vivo assessment using a rat cranial defect model. Drug-loaded scaffolds showed controlled release of siponimod, -cytocompatibility with endothelial and osteoblast cells in vitro, and furthermore, showed that released siponimod stimulated osteoblast differentiation and endothelial cell migration. The in vivo cranial defect repair study showed regeneration was occurring in the defect, although there was no significant difference in the extent of mineralisation between scaffold experimental groups. To our knowledge, this is the first study investigating siponimod in bone regeneration. In vitro studies confirm a positive impact on key cells involved in bone regeneration, however, the scaffolds did not result in significant repair of critical cranial defects.

Mesoporous Bioactive Glass Scaffold Delivers Salvianolic Acid B to Promote Bone Regeneration in a Rat Cranial Defect Model

Mesoporous Bioactive Glass (MBG) has been widely studied because of its excellent histocompatibility and degradability. However, due to the lack of good osteoinductive activity, the pure MBG scaffold is not effective in repairing large-scale bone defects. To observe the repair effect of MBG scaffolds delivering Salvianolic acid B (SB) on critical bone defects in rats. In this study, MBG scaffolds were used as delivery vehicle. SB, a small molecular active drug with good osteogenic differentiation ability, was loaded into the MBG scaffolds at low, medium and high doses. The effect of SB released from the MBG scaffolds on osteogenic differentiation of rat Bone Marrow Mesenchymal Stem Cells (rBMSCs) was investigated using alkaline phosphatase staining, alizarin red staining and real-time quantitative polymerase chain reaction. Moreover, 8 weeks after implantation of the scaffolds, the bone regeneration was evaluated by micro- CT, sequential fluorescence labeling and histological staining analysis. The in vitro results showed that different doses of SB had similar release rate from scaffolds and could be released from scaffolds continuously. The middle dose (MBG/MSB) and high dose (MBG/HSB) groups significantly promoted the osteogenic differentiation of rBMSCs when compared with a low dose (MBG/LSB) group. Moreover, SB produced significant increases in newly formed bone of calvarial bone defects in rats. It is concluded that the use of MBG scffolds delivering SB is an effective strategy for the treatment of bone defects.

A Novel 3D-bioprinted Porous Nano Attapulgite Scaffolds with Good Performance for Bone Regeneration.

BackgroundNatural clay nanomaterials are an emerging class of biomaterial with great potential for tissue engineering and regenerative medicine applications, most notably for osteogenesis.Materials and MethodsHerein, for the first time, novel tissue engineering scaffolds were prepared by 3D bioprinter using nontoxic and bioactive natural attapulgite (ATP) nanorods as starting materials, with polyvinyl alcohol as binder, and then sintered to obtain final scaffolds. The microscopic morphology and structure of ATP particles and scaffolds were observed by transmission electron microscope and scanning electron microscope. In vitro biocompatibility and osteogenesis with osteogenic precursor cell (hBMSCs) were assayed using MTT method, Live/Dead cell staining, alizarin red staining and RT-PCR. In vivo bone regeneration was evaluated with micro-CT and histology analysis in rat cranium defect model.ResultsWe successfully printed a novel porous nano-ATP scaffold designed with inner channels with a dimension of 500 µm and wall structures with a thickness of 330 µm. The porosity of current 3D-printed scaffolds ranges from 75% to 82% and the longitudinal compressive strength was up to 4.32±0.52 MPa. We found firstly that nano-ATP scaffolds with excellent biocompatibility for hBMSCscould upregulate the expression of osteogenesis-related genes bmp2 and runx2 and calcium deposits in vitro. Interestingly, micro-CT and histology analysis revealed abundant newly formed bone was observed along the defect margin, even above and within the 3D bioprinted porous ATP scaffolds in a rat cranial defect model. Furthermore, histology analysis demonstrated that bone was formed directly following a process similar to membranous ossification without any intermediate cartilage formation and that many newly formed blood vessels are within the pores of 3D-printed scaffolds at four and eight weeks.ConclusionThese results suggest that the 3D-printed porous nano-ATP scaffolds are promising candidates for bone tissue engineering by osteogenesis and angiogenesis.

Advanced reconfigurable scaffolds fabricated by 4D printing for treating critical-size bone defects of irregular shapes

While scaffold-based tissue engineering has been widely used to treat bone critical-size defects, challenges such as implantation of scaffolds in defects with irregular shapes and implantation of scaffolds through minimally invasive surgery remain in the tissue engineering field. Customized bioactive bone tissue engineering scaffolds with reconfigurable capability for both easy scaffold implantation and perfect shape fitting in irregularly shaped bone defects are therefore needed. Herein, applying 4D printing, photothermal-responsive shape memory bone tissue engineering scaffolds are constructed by incorporating black phosphorus nanosheets and osteogenic peptide into β-tricalcium phosphate/poly(lactic acid-co-trimethylene carbonate) (TCP/P(DLLA-TMC)) nanocomposite scaffolds. When near-infrared irradiation is applied to customized scaffolds on-demand, scaffold temperature rapidly increases to 45 °C, enabling scaffold shape reconfiguration for easy scaffold implantation and precise fitting in irregular bone defects. Once the implantation is finished, scaffold temperature rapidly decreases to 37 °C and scaffolds display mechanical properties comparable to those of human cancellous bone. The improved osteogenesis in bone defect sites is then initiated through pulsed peptide release from scaffolds. Compact integration of reconfigurable scaffolds in rat cranial bone defects and improved new bone formation are demonstrated through micro-computed tomography and histochemical analyses. This study shows a facile method to clinically treat bone defects of irregular shapes.

Delivering Proangiogenic Factors from 3D-Printed Polycaprolactone Scaffolds for Vascularized Bone Regeneration.

Natural bone is a highly vascularized tissue that relies on the vasculature for blood and nutrients supply to maintain skeletal integrity. Inadequacy of neovascularization may compromise the tissue ingrowth to the implanted scaffolds, and eventually results in failure for the repair. To tackle this issue and enhance self-vascularized bone regeneration, herein a 3D biomimetic selective lasersintering (SLS) derived scaffold, with an angiogenic growth factor immobilized on its surface, that can be released in a controlled manner is proposed. To this end, a porous polycaprolactone/hydroxyapatite (PCL/HA) scaffold is prepared via the SLS technique, which is further modified with vascular endothelial growth factor (VEGF) by coprecipitation with apatite. The resultant scaffold (PCL/HA/VEGF) has an excellent cytocompatibility, and subcutaneous implantation experiment shows that the VEGF-loaded scaffold significantly enhances the blood vessel formation compared with the VEGF-free control. It is further demonstrated that the PCL/HA/VEGF scaffold is able to enhance the in vivo bone regeneration in a rat cranial defect model. Taken together, the current study provides not only a feasible and promising scaffold candidate to enhance the vascularized bone regeneration, but also a general strategy to overcome the inadequate vascularization issue for the repair of other tissue and organs.

Repair of rat cranial bone defect by using amniotic fluid-derived mesenchymal stem cells in polycaprolactone fibrous scaffolds and platelet-rich plasma

Introduction: Tissue regenerative medicine strategies, as a promising alternative has become of major interest to the reconstruction of critical size bone defects. This study evaluated the effects of the simultaneous application of polycaprolactone (PCL), amniotic fluid mesenchymal stem cells (AF-MSCs) and platelet-rich plasma (PRP) on the repair of rat cranial bone defects. Methods: The AF-MSCs were isolated at the end of the second week of pregnancy in rats. PRP obtained from rat blood and the random PCL fibrous scaffolds were prepared using the electrospinning method. Circular full thickness (5 mm) bone defects were developed on both sides of the parietal bones (animal number=24) and the scaffolds containing AF-MSCs and PRP were implanted in the right lesions. Thereafter, after eight weeks the histological and immunohistochemistry studies were performed to evaluate the bone formation and collagen type I expression. Results: The spindle-shaped mesenchymal stem cells were isolated and the electron microscope images indicated the preparation of a random PCL scaffold. Immunohistochemical findings showed that collagen type I was expressed by AF-MSCs cultured on the scaffold. The results of hematoxylin and eosin (H&E) staining indicated the formation of blood vessels in the presence of PRP. Additionally, immunofluorescence findings suggested that PRP had a positive effect on collagen type I expression. Conclusion: The simultaneous application of fibrous scaffold + AF-MSCs + PRP has positive effects on bone regeneration. This study showed that PRP can affect the formation of new blood vessels in the scaffold transplanted in the bone defect.

Mitochondria transfer enhances proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cell and promotes bone defect healing

BackgroundBone marrow-derived mesenchymal stem cell (BMSC) transplantation is considered a promising therapeutic approach for bone defect repair. However, during the transplantation procedure, the functions and viability of BMSCs may be impaired due to extended durations of in vitro culture, aging, and disease conditions of patients. Inspired by spontaneous intercellular mitochondria transfer that naturally occurs within injured tissues to rescue cellular or tissue function, we investigated whether artificial mitochondria transfer into pre-transplant BMSCs in vitro could improve cellular function and enhance their therapeutic effects on bone defect repair in situ.MethodsMitochondria were isolated from donor BMSCs and transferred into recipient BMSCs of the same batch and passage. Subsequently, changes in proliferative capacity and cell senescence were evaluated by live cell imaging, Cell Counting Kit-8 assay, cell cycle analysis, Ki67 staining, qPCR and Western blot analysis of c-Myc expression, and β-galactosidase staining. Migration ability was evaluated by the transwell migration assay, wound scratch healing, and cell motility tests. Alkaline phosphatase (ALP) staining, Alizarin Red staining, and combined with qPCR and Western blot analyses of Runx2 and BMP2 were performed to elucidate the effects of mitochondria transfer on the osteogenic potential of BMSCs in vitro. After that, in vivo experiments were performed by transplanting mitochondria-recipient BMSCs into a rat cranial critical-size bone defect model. Micro CT scanning and histological analysis were conducted at 4 and 8 weeks after transplantation to evaluate osteogenesis in situ. Finally, in order to establish the correlation between cellular behavioral changes and aerobic metabolism, OXPHOS (oxidative phosphorylation) and ATP production were assessed and inhibition of aerobic respiration by oligomycin was performed.ResultsMitochondria-recipient BMSCs exhibited significantly enhanced proliferation and migration, and increased osteogenesis upon osteogenic induction. The in vivo results showed more new bone formation after transplantation of mitochondria-recipient BMSCs in situ. Increased OXPHOS activity and ATP production were observed, which upon inhibition by oligomycin attenuated the enhancement of proliferation, migration, and osteogenic differentiation induced by mitochondria transfer.ConclusionsMitochondria transfer is a feasible technique to enhance BMSC function in vitro and promote bone defect repair in situ through the upregulation of aerobic metabolism. The results indicated that mitochondria transfer may be a novel promising technique for optimizing stem cell therapeutic function.

Osteogenic growth peptide-loaded 3D-printed PCL scaffolds for the promotion of osteogenesis through the ERK pathway

This study aims to improve the osteogenic differentiation in vitro and bone formation in vivo through polycaprolactone (PCL) scaffolds modified with the osteogenic growth peptide (OGP). Three-dimensional (3D)-printed PCL scaffolds were designed with computer-aided design (CAD) software and fabricated by 3D layer-by-layer fused deposition. After the scaffold surfaces were modified by surface amination, the OGP was successfully loaded onto the scaffolds using a electrostatic self-assembly method. The effects of these scaffolds on the biocompatibility and osteogenic differentiation of rat bone marrow-derived stem cells (BMSCs) were studied in vitro. The modification did not influence the adhesion or proliferation, but had a notable osteogenic effect in vitro. This may be attributed to the activation of the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) signalling pathway. Based on the results obtained after implantation into a rat cranial defect model, the PCL/OGP scaffolds notably promoted bone formation and accelerated mineralisation. The innovative modification method introduced in this work, will improve the application of PCL scaffolds in tissue engineering.