3D printed PCL/SrHA scaffold for enhanced bone regeneration

A 3D printed polylactic acid-Baghdadite nanocomposite scaffold coated with microporous chitosan-VEGF for bone regeneration applications

As a biodegradable polymer thin film, silk fibroin/chitosan composite film overcomes the defects of pure silk fibroin and chitosan films, respectively, and shows remarkable biocompatibility, appropriate hydrophilicity and mechanical properties. Silk fibroin/chitosan thin film can be used not only as metal implant coating for bone injury repair, but also as tissue engineering scaffold for skin, cornea, adipose, and other soft tissue injury repair. However, the biocompatibility of silk fibroin/chitosan thin film for mesenchymal stem cells, a kind of important seed cell of tissue engineering and regenerative medicine, is rarely reported. In this study, silk fibroin/chitosan film was prepared by solvent casting method, and the rat bone marrow-derived mesenchymal stem cells were cultured on the silk fibroin/chitosan thin film. Osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells were induced, respectively. The proliferation ability, osteogenic and adipogenic differentiation abilities of rat bone marrow-derived mesenchymal stem cells were systematically compared between silk fibroin/chitosan thin film and polystyrene tissue culture plates. The results showed that silk fibroin/chitosan thin film not only provided a comparable environment for the growth and proliferation of rat bone marrow-derived mesenchymal stem cells but also promoted their osteogenic and adipogenic differentiation. This work provided information of rat bone marrow-derived mesenchymal stem cells behavior on silk fibroin/chitosan thin film and extended the application of silk fibroin/chitosan thin film. Based on the results, we suggested that the silk fibroin/chitosan thin film could be a promising material for tissue engineering of bone, cartilage, adipose, and skin.

Repairing irregular-shaped bone defects poses enormous challenges. Scaffolds that can fully fit the defect site and simultaneously induce osteogenesis and angiogenesis hold great promise for bone defect healing. This study aimed to produce superelastic organic/inorganic composite aerogel scaffolds by blending silica nanofibers (SiO2 ) and poly (lactic acid)/gelatin (PLA/gel) nanofibers; the content of SiO2 nanofibers is varied from 0-60 wt% (e.g., PLA/gel, PLA/gel/SiO2 -L, PLA/gel/SiO2 -M, and PLA/gel/SiO2 -H for 0%, 20%, 40%, and 60% of SiO2 nanofibers, respectively) to produce a range of scaffolds. The PLA/gel/SiO2 -M scaffold has excellent elasticity and good mechanical properties. In vitro experiments demonstrate that the silicon ions released from PLA/gel/SiO2 -M scaffolds promote the differentiation of rat bone marrow-derived mesenchymal stem cells into osteoblasts, enhancing alkaline phosphatase activity and bone-related genes expressions. The released silicon ions also promote the proliferation of human umbilical vein endothelial cells and the expression of vascular endothelial growth factors, thereby promoting angiogenesis. The assessment of these scaffolds in a calvarial defect model in rats shows good potential of PLA/gel/SiO2 -M to induce bone regeneration as well as promote osteogenesis and angiogenesis. Overall, these organic/inorganic composite scaffolds have good biological activity, which may have broad applications for tissue engineering.

Development of bioactive scaffolds with controllable architecture and high osteogenic capability for bone tissue engineering is hotly pursued. In this study, three-dimensional (3D) mesoporous bioactive glass (MBG) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds with well-defined pore structures and high compressive strength (∼5-12 MPa) were synthesized by a 3D printing technique. Compared to reported polymer-bonded MBG scaffolds, the incorporation of the biocompatible PHBHHx polymer as a particle binder enhanced their bioactive and osteogenic properties, including fast apatite-forming ability, and promoted human bone marrow-derived mesenchymal stem cell (hBMSC) adhesion, proliferation, alkaline phosphatase (ALP) activity and bone-related gene expression. Furthermore, MBG/PHBHHx composite scaffolds were explored to repair critical-size rat calvarial defects. The results showed that MBG/PHBHHx composite scaffolds exhibited a controlled degradation rate and more significant potential to stabilize the pH environment with increasing PHBHHx ratio. At 8 weeks post-implantation, MBG/PHBHHx scaffolds were demonstrated to stimulate bone regeneration in the calvarial defects and have largely repaired them through analysis of micro-CT, sequential fluorescent labeling and histology. These results lay a potential framework for future study by using modified MBG/PHBHHx-based functional scaffolds to improve the osteogenic activity and bone defect restoration.

Oxidative stress hurts the survival of transplanted mesenchymal stem cells (MSCs). Lipopolysaccharide (LPS) preconditioning inhibits apoptotic death in MSCs. Also, Lovastatin's protective effect was reported on MSCs. Here, we investigated the potential of LPS and Lovastatin combination therapy on the survival and proliferation of MSCs. MSCs harvested from adult rats (240-260 g) femur and tibia bone marrow. Third passage MSCs were divided into 6 groups control group, LPS, LPS + Lovastatin (10 and 15 µM), and Lovastatin (10 and 15 µM). Cell survival and proliferation were assessed using an MTT assay 24 h after LPS, Lovastatin, or LPS + Lovastatin treatment. Also, Malondialdehyde (MDA) as a lipid peroxidation marker and antioxidant enzymes such as Glutathione peroxidase (GPX) and Superoxide dismutase (SOD) activity levels evaluated. Finally, the expression level of tumor protein P53 (P53) and octamer-binding transcription factor 4 (OCT4) genes were measured by qRT-PCR test. Lovastatin 10 μM potentiated proliferation and survival of MSCs. It can increase the activity of GPX and SOD. 10 µM Lovastatin could not affect MDA amounts but decreased the expression levels of P53 and Oct4 significantly. Nevertheless, treatment with LPS reduced the survival and proliferation of MSCs, along with a significant reduction in GPX activity. LPS + Lovastatin could increase SOD activity, however, GPX enzyme activity and MSCs proliferation did not change so, and it was not effective. We propose Lovastatin at the dose of 10 µM as a suitable combination agent to increase the survival and proliferation of MSCs in oxidative stress conditions.

Bone is the rigid tissue that constitutes the skeleton. The material for bone regeneration has to provide the mechanical stability by maintaining the mechanical loads both in the rest conditions and during the body movements. Bone is dynamic tissue constantly reshaped by the action of cells (osteoblasts and osteoclasts). This activity is normally enough to heal bone injuries; however, in several conditions, when bone is subjected to fatal damages, self-renewal is difficult, if not even impossible, and a medical treatment is required. The transplantation of a biomaterial is one of the common surgical procedures to overcome critical injuries. In this study, we exploited the effect of the use of different sources of demineralized bone powder (DBP) in combination with gellan gum (GG) to form a GG-DBP hydrogel scaffold with the purpose of regenerating the bone tissue. DBP was extracted from the femurs of two typologies of Gallus gallus domesticus (the Yeonsan Ogye, a traditional and rare black chicken from Korea, and the Cornish cross, the most common breeds for industrial meat production) and the Pekin duck. The composite scaffold has been tested both in vitro and in vivo. In vitro studies using rat bone marrow-derived mesenchymal stem cells (rBMSCs) confirmed the cellular suitability of bone-specific gene expression for seeded GG-DBP scaffolds, differentiation capacity, and marked upregulation. The scaffold containing a DBP derived from the Yeonsan Ogye (YO) bone showed higher levels of cell proliferation and osteogenic differentiation in comparison with the scaffold with the DBP obtained from the other studied sources. These results have been related with the higher amount of melanin, studied by fluorescence, of the YO DBP compared to Cornish cross and Pekin duck. Overall, this study clearly shows the use of YO DBP as a promising material in bone tissue regeneration.

Three-dimensional printed sodium alginate clay nanotube composite scaffold for bone regeneration

Tissue Engineering Designs for the Future: New Logics, Old Molecules

Tissue-engineered bone material is one of the effective methods to repair bone defects, but the application is restricted in clinical because of the lack of excellent scaffolds that can induce bone regeneration as well as the difficulty in making scaffolds with personalized structures. 3D printing is an emerging technology that can fabricate bespoke 3D scaffolds with precise structure. However, it is challenging to develop the scaffold materials with excellent printability, osteogenesis ability, and mechanical strength. In this study, graphene oxide (GO), attapulgite (ATP), type I collagen (Col I) and polyvinyl alcohol were used as raw materials to prepare composite scaffolds via 3D bioprinting. The composite materials showed excellent printability. The microcosmic architecture and properties was characterized by scanning electron microscopy, Fourier transform infrared and thermal gravimetric analyzer, respectively. To verify the biocompatibility of the scaffolds, the viability, proliferation and osteogenic differentiation of Bone Marrow Stromal Cells (BMSCs) on the scaffolds were assessed by CCK-8, Live/Dead staining and Real-time PCR in vitro. The composited scaffolds were then implanted into the skull defects on rat for bone regeneration. Hematoxylin-eosin staining, Masson staining and immunohistochemistry staining were carried out in vivo to evaluate the regeneration of bone tissue.The results showed that GO/ATP/COL scaffolds have been demonstrated to possess controlled porosity, water absorption, biodegradability and good apatite-mineralization ability. The scaffold consisting of 0.5% GO/ATP/COL have excellent biocompatibility and was able to promote the growth, proliferation and osteogenic differentiation of mouse BMSCs in vitro. Furthermore, the 0.5% GO/ATP/COL scaffolds were also able to promote bone regeneration of in rat skull defects. Our results illustrated that the 3D printed GO/ATP/COL composite scaffolds have good mechanical properties, excellent cytocompatibility for enhanced mouse BMSCs adhesion, proliferation, and osteogenic differentiation. All these advantages made it potential as a promising biomaterial for osteogenic reconstruction.

Stem cell derived small extracellular vesicles (sEVs) have emerged as promising nanomaterials for the repair of bone defects. However, low retention of sEVs affects their therapeutic effects. Clinically used natural substitute inorganic bovine bone mineral (Bio-Oss) bone powder lacks high compactibility and efficient osteo-inductivity that limit its clinical application in repairing large bone defects. In this study, a poly ethylene glycol/hyaluronic acid (PEG/HA) hydrogel was used to stabilize Bio-Oss and incorporate rat bone marrow stem cell-derived sEVs (rBMSCs-sEVs) to engineer a PEG/HA-Bio-Oss (PEG/HA-Bio) composite scaffold. Encapsulation and sustained release of sEVs in hydrogel scaffold can enhance the retention of sEVs in targeted area, achieving long-lasting repair effect. Meanwhile, synergistic administration of sEVs and Bio-Oss in cranial defect can improve therapeutic effects. The PEG/HA-Bio composite scaffold showed good mechanical properties and biocompatibility, supporting the growth of rBMSCs. Furthermore, sEVs enhancedin vitrocell proliferation and osteogenic differentiation of rBMSCs. Implantation of sEVs/PEG/HA-Bio in rat cranial defect model promotedin vivobone regeneration, suggesting the great potential of sEVs/PEG/HA-Bio composite scaffold for bone repair and regeneration. Overall, this work provides a strategy of combining hydrogel composite scaffold systems and stem cell-derived sEVs for the application of tissue engineering repair.

目的 研究提高和阻断端粒酶活性对大鼠骨髓间充质干细胞(BMSCs)增殖及分化的影响.方法采用直接贴壁法分离、培养SD大鼠BMSCs,流式细胞仪检测BMSCs表面标记.阳离子脂质体介导hTERT正、反义表达载体转染BMSCs,RT-PCR和免疫组化方法检测TERT表达,并对转染前后BMSCs的生长增殖能力、神经细胞方向的分化能力和大鼠颅内移植存活情况进行研究.结果大鼠BMSCs表面CD29、CD44、CD90阳性,CD31、CD34、CD45阴性,转染正义hTERT后BMSCs增殖速度明显提高,而其向神经元和星形胶质细胞诱导分化及异体海马移植后的存活能力未受影响,转染反义hTERT后BMSCs增殖速度减慢,5～6周内死亡. 结论BMSCs存在端粒酶低表达,提高或阻断端粒酶活性对BMSCs的增殖分别起到促进或抑制作用。

The application of a three-dimensional (3D)-printed biological functional scaffold in the repair of bone defects is a promising strategy. In this study, strontium-containing hydroxyapatite (SrHA) powder was synthesized by the hydrothermal method, and then poly(ε-caprolactone) (PCL)/HA and PCL/SrHA composite scaffolds were prepared by the high-temperature melt extrusion 3D printing technology. The basic physical and chemical properties, in vitro biological properties, osteogenesis, and angiogenesis abilities of the scaffold were studied. The results showed that HA and SrHA were uniformly embedded in the composite scaffold, and the scaffold exhibited a 3D interconnected porous structure and rough microsurface. The in vitro release curve showed that Sr2+ and Ca2+ were continuously released from the PCL/SrHA scaffold. In order to verify the performance of the composite scaffold in bone regeneration, the proliferation and osteogenic differentiation of mouse embryonic osteoblasts (MC3T3E1) grown on the scaffold were evaluated. The experimental results showed that the incorporation of SrHA significantly promoted cell proliferation. Compared with the PCL/HA scaffold, the PCL/SrHA scaffold could better promote cell osteogenic differentiation. Deferoxamine (DFO) was loaded on the surface of the PCL/SrHA scaffold. By studying the proliferation, angiogenesis, and expression of osteogenesis and angiogenesis-related genes of human umbilical vein endothelial cells (HUVECs) on PCL/SrHA@DFO scaffold, it was verified that DFO had the ability to promote angiogenesis. It could induce angiogenesis in vitro in combination with Sr2+. Therefore, we believe that the composite scaffold has potential application prospects in the field of bone tissue engineering.

Osteogenic peptides have been reported as highly effective in directing mesenchymal stem cell osteogenic differentiation in vitro and bone formation in vivo. Therefore, developing novel biomaterials for the controlled delivery of osteogenic peptides in scaffolds without lowering the peptide’s biological activity is highly desirable. To repair a critical-sized bone defect to efficiently achieve personalized bone regeneration, a novel bioactive poly(lactic-co-glycolic acid) (PLGA)/β-tricalcium phosphate (β-TCP) composite scaffold, in which graphene oxide (GO) and bone morphogenetic protein (BMP)-2-like peptide were loaded in situ (PTG/P), was produced by an original cryogenic 3D printing method. The scaffolds were mechanically comparable to human cancellous bone and hierarchically porous. The incorporation of GO further improved the scaffold wettability and mechanical strength. The in situ loaded peptides retained a high level of biological activity for an extended time, and the loading of GO in the scaffold further tuned the peptide release so that it was more sustained. Our in vitro study showed that the PTG/P scaffold promoted rat bone marrow-derived mesenchymal stem cell ingrowth into the scaffold and enhanced osteogenic differentiation. Moreover, the in vivo study indicated that the novel PTG/P scaffold with sustained delivery of the peptide could significantly promote bone regeneration in a critical bone defect. Thus, the novel bioactive PTG/P scaffold with a customized shape, improved mechanical strength, sustainable peptide delivery, and excellent osteogenic ability has great potential in bone tissue regeneration.

Osteogenesis and angiogenesis induced by porous β-CaSiO3/PDLGA composite scaffold via activation of AMPK/ERK1/2 and PI3K/Akt pathways

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca2+‐activated K+ channel, KCa3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the KCa3.1 channel expression and its role in rat bone marrow‐derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up‐regulated the KCa3.1 channel expression and pharmacological or genetic inhibition of the KCa3.1 channel strongly suppressed stretch‐induced increase in cell proliferation and cell cycle progression. These results support that the KCa3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.