The MnO2/GelMA Composite Hydrogels Improve the ROS Microenvironment of Annulus Fibrosus Cells by Promoting the Antioxidant and Autophagy through the SIRT1/NRF2 Pathway.

Intervertebral disc degeneration (IVDD) is a worldwide disease that causes low back pain and reduces quality of life. Biotherapeutic strategies based on tissue engineering alternatives, such as intervertebral disc scaffolds, supplemented by drug-targeted therapy have brought new hope for IVDD. In this study, to explore the role and mechanism of MnO2/GelMA composite hydrogels in alleviating IVDD, we prepared composite hydrogels with MnO2 and methacrylate gelatin (GelMA) and characterized them using compression testing and transmission electron microscopy (TEM). Annulus fibrosus cells (AFCs) were cultured in the composite hydrogels to verify biocompatibility by live/dead and cytoskeleton staining. Cell viability assays and a reactive oxygen species (ROS) probe were used to analyze the protective effect of the composite hydrogels under oxidative damage. To explore the mechanism of improving the microenvironment, we detected the expression levels of antioxidant and autophagy-related genes and proteins by qPCR and Western blotting. We found that the MnO2/GelMA composite hydrogels exhibited excellent biocompatibility and a porous structure, which promoted cell proliferation. The addition of MnO2 nanoparticles to GelMA cleared ROS in AFCs and induced the expression of antioxidant and cellular autophagy through the common SIRT1/NRF2 pathway. Therefore, the MnO2/GelMA composite hydrogels, which can improve the disc microenvironment through scavenging intracellular ROS and resisting oxidative damage, have great application prospects in the treatment of IVDD.

Biomimetic triphasic silk fibroin scaffolds seeded with tendon-derived stem cells for tendon-bone junction regeneration.

The regeneration of tendon and bone junctions (TBJs), a fibrocartilage transition zone between tendons and bones, is a challenge due to the special triphasic structure. In our study, a silk fibroin (SF)-based triphasic scaffold consisting of aligned type I collagen (Col I), transforming growth factor ß (TGF-ß), and hydroxyapatite (HA) was fabricated to mimic the compositional gradient feature of the native tendon-bone architecture. Rat tendon-derived stem cells (rTDSCs) were loaded on the triphasic SF scaffold, and the high cell viability suggested that the scaffold presents good biocompatibility. Meanwhile, increased expressions of tenogenic-, chondrogenic-, and osteogenic-related genes in the TBJs were observed. The in vivo studies of the rTDSC-seeded scaffold in a rat TBJ rupture model showed tendon tissue regeneration with a clear transition zone within 8 weeks of implantation. These results indicated that the biomimetic triphasic SF scaffolds seeded with rTDSCs have great potential to be applied in TBJ regeneration.

High-Strength Smart Microneedles with "Offensive and Defensive" Effects for Intervertebral Disc Repair.

Intervertebral disc degeneration (IVDD) is a global public health issue. The injury of annulus fibrosus (AF) caused by acupuncture or discectomy can trigger IVDD again. However, there is currently no suitable method for treating AF injury. In this study, the high-strength smart microneedles (MNs) which can penetrate the AF tissue through a local and minimally invasive method, and achieve remote control of speeded-up release of the drug and hyperthermia by the Near Infrared is developed. The PDA/GelMA composite MNs loaded with diclofenac sodium are designed to extracellularly "offend" the inflammatory microenvironment and mitigate damage to cells, and intracellularly increase the level of cytoprotective heat shock proteins to enhance the defense against the hostile microenvironment, achieving "offensive and defensive" effects. In vitro experiments demonstrate that the synergistic treatment of photothermal therapy and anti-inflammation effectively reduces inflammation, inhibits cell apoptosis, and promotes the synthesis of the extracellular matrix (ECM). In vivo experiments show that the MNs mitigate the inflammatory response, promote ECM deposition, reduce the level of apoptosis, and restore the biomechanical properties of the intervertebral disc (IVD) in rats. Overall, this high-strength smart MNs display promising "offensive and defensive" effects that can provide a new strategy for IVD repair.

Engineered biomimetic micro/nano-materials for tissue regeneration.

The incidence of tissue and organ damage caused by various diseases is increasing worldwide. Tissue engineering is a promising strategy of tackling this problem because of its potential to regenerate or replace damaged tissues and organs. The biochemical and biophysical cues of biomaterials can stimulate and induce biological activities such as cell adhesion, proliferation and differentiation, and ultimately achieve tissue repair and regeneration. Micro/nano materials are a special type of biomaterial that can mimic the microstructure of tissues on a microscopic scale due to its precise construction, further providing scaffolds with specific three-dimensional structures to guide the activities of cells. The study and application of biomimetic micro/nano-materials have greatly promoted the development of tissue engineering. This review aims to provide an overview of the different types of micro/nanomaterials, their preparation methods and their application in tissue regeneration.

Silk Fibroin/Wool Keratin Composite Scaffold with Hierarchical Fibrous and Porous Structure.

The present study describes a silk microfiber reinforced meniscus scaffold (SMRMS) with hierarchical fibrous and porous structure made from silk fibroin (SF) and wool keratin (WK) using electrospinning and freeze-drying technology. This study focuses on the morphology, secondary structure, mechanical properties, and water absorption properties of the scaffold. The cytotoxicity and biocompatibility of SMRMS are assessed in vivo and in vitro. The scaffold shows hierarchical fibrous and porous structure, hierarchical pore size distribution (ranges from 50 to 650 µm), robust mechanical properties (compression strength can reach at 2.8 MPa), and stable biodegradability. A positive growth condition revealed by in vitro cytotoxicity testing indicates that the scaffold is not hazardous to cells. In vivo assessments of biocompatibility reveal that only a mild inflammatory reaction is present in implanted rat tissue. Meniscal scaffold made of SF/WK composite shows a potential application prospect in the meniscal repair engineering field with its development.

Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration.

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades. However, efficient harvest and handling of cell sheets remain challenging, including insufficient extracellular matrix content and poor mechanical strength. Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types. However, currently, there are no effective ways to apply mechanical loading to cell sheets. In this study, we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide) (PNIPAAm) to poly(dimethylsiloxane) (PDMS) surfaces. The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting. Subsequently, MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates. Upon maturation, the cell sheets were harvested by lowering the temperature. We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning. Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated. After implantation into the critical-sized calvarial defects of mice, the mechanically conditioned cell sheets significantly promoted new bone formation. Findings from this study reveal that thermo-responsive elastomer, together with mechanical conditioning, can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.

Transformation of arginine into zero-dimensional nanomaterial endows the material with antibacterial and osteoinductive activity.

Implant-associated infection is a major threat affecting the success of orthopedic surgeries. Although various materials scavenge bacteria by generating reactive oxygen species (ROS), the intrinsic inability of ROS to distinguish bacteria from cells notably limits the therapeutic effects. Here, we found that the arginine carbon dots (Arg-CDs) that were transformed from arginine exhibited supreme antibacterial and osteoinductive activity. We further designed the Schiff base bond between Arg-CDs and aldehyde hyaluronic acid/gelatin methacryloyl (HG) hydrogel to release Arg-CDs in response to the acidic bone injury microenvironment. The free Arg-CDs could selectively kill bacteria by generating excessive ROS. Furthermore, the Arg-CD-loaded HG composite hydrogel showed excellent osteoinductive activity through inducing the M2 polarization of macrophages by up-regulating interleukin-10 (Il10) expression. Together, our findings revealed that transformation of the arginine into zero-dimensional Arg-CDs could endow the material with exceptional antibacterial and osteoinductive activity, favoring the regeneration of infectious bone.

Building Osteogenic Microenvironments with a Double-Network Composite Hydrogel for Bone Repair.

The critical factor determining the in vivo effect of bone repair materials is the microenvironment, which greatly depends on their abilities to promote vascularization and bone formation. However, implant materials are far from ideal candidates for guiding bone regeneration due to their deficient angiogenic and osteogenic microenvironments. Herein, a double-network composite hydrogel combining vascular endothelial growth factor (VEGF)-mimetic peptide with hydroxyapatite (HA) precursor was developed to build an osteogenic microenvironment for bone repair. The hydrogel was prepared by mixing acrylated ß-cyclodextrins and octacalcium phosphate (OCP), an HA precursor, with gelatin solution, followed by ultraviolet photo-crosslinking. To improve the angiogenic potential of the hydrogel, QK, a VEGF-mimicking peptide, was loaded in acrylated ß-cyclodextrins. The QK-loaded hydrogel promoted tube formation of human umbilical vein endothelial cells and upregulated the expression of angiogenesis-related genes, such as Flt1, Kdr, and VEGF, in bone marrow mesenchymal stem cells. Moreover, QK could recruit bone marrow mesenchymal stem cells. Furthermore, OCP in the composite hydrogel could be transformed into HA and release calcium ions facilitating bone regeneration. The double-network composite hydrogel integrated QK and OCP showed obvious osteoinductive activity. The results of animal experiments showed that the composite hydrogel enhanced bone regeneration in skull defects of rats, due to perfect synergistic effects of QK and OCP on vascularized bone regeneration. In summary, improving the angiogenic and osteogenic microenvironments by our double-network composite hydrogel shows promising prospects for bone repair.

Targeting Endogenous Reactive Oxygen Species Removal and Regulating Regenerative Microenvironment at Annulus Fibrosus Defects Promote Tissue Repair.

The excessive reactive oxygen species (ROS) level, inflammation, and weak tissue regeneration ability after annulus fibrosus (AF) injury constitute an unfavorable microenvironment for AF repair. AF integrity is crucial for preventing disc herniation after discectomy; however, there is no effective way to repair the AF. Herein, a composite hydrogel integrating properties of antioxidant, anti-inflammation, and recruitment of AF cells is developed through adding mesoporous silica nanoparticles modified by ceria and transforming growth factor ß3 (TGF-ß3) to the hydrogels. The nanoparticle loaded gelatin methacrylate/hyaluronic acid methacrylate composite hydrogels eliminate ROS and induce anti-inflammatory M2 type macrophage polarization. The released TGF-ß3 not only plays a role in recruiting AF cells but is also responsible for promoting extracellular matrix secretion. The composite hydrogels can be solidified in situ in the defect area to effectively repair AF in rats. The strategies targeting endogenous ROS removal and improving the regenerative microenvironment by the nanoparticle-loaded composite hydrogels have potential applications in AF repair and intervertebral disc herniation prevention.

TGF-ß1-supplemented decellularized annulus fibrosus matrix hydrogels promote annulus fibrosus repair.

Annulus fibrosus (AF) repair remains a challenge because of its limited self-healing ability. Endogenous repair strategies combining scaffolds and growth factors show great promise in AF repair. Although the unique and beneficial characteristics of decellularized extracellular matrix (ECM) in tissue repair have been demonstrated, the poor mechanical property of ECM hydrogels largely hinders their applications in tissue regeneration. In the present study, we combined polyethylene glycol diacrylate (PEGDA) and decellularized annulus fibrosus matrix (DAFM) to develop an injectable, photocurable hydrogel for AF repair. We found that the addition of PEGDA markedly improved the mechanical strength of DAFM hydrogels while maintaining their porous structure. Transforming growth factor-ß1 (TGF-ß1) was further incorporated into PEGDA/DAFM hydrogels, and it could be continuously released from the hydrogel. The in vitro experiments showed that TGF-ß1 facilitated the migration of AF cells. Furthermore, PEGDA/DAFM/TGF-ß1 hydrogels supported the adhesion, proliferation, and increased ECM production of AF cells. In vivo repair performance of the hydrogels was assessed using a rat AF defect model. The results showed that the implantation of PEGDA/DAFM/TGF-ß1 hydrogels effectively sealed the AF defect, prevented nucleus pulposus atrophy, retained disc height, and partially restored the biomechanical properties of disc. In addition, the implanted hydrogel was infiltrated by cells resembling AF cells and well integrated with adjacent AF tissue. In summary, findings from this study indicate that TGF-ß1-supplemented DAFM hydrogels hold promise for AF repair.

Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds.

There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue. Three-dimensional (3D) printing offers a method of fabricating complex anatomical features of clinically relevant sizes. However, the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging. This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions. The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional (2D). The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices, which were arrayed. These 2D slices with each layer of a defined pattern were laser cut, and then successfully assembled with varying thicknesses of 100 µm or 200 µm. It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions, where the clinically relevant sizes ranging from a simple cube of 20 mm dimension, to a more complex, 50 mm-tall human ears were created. In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure. The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice, where a range of hole diameters from 200 µm to 500 µm were laser cut in an array where cell confluence values of at least 85% were found at three weeks. Cells were also seeded onto a simpler stacked construct, albeit made with micromachined poly fibre mesh, where cells can be found to migrate through the stack better with collagen as bioadhesives. This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future.

High-resolution 3D printing of angle-ply annulus fibrosus scaffolds for intervertebral disc regeneration.

Intervertebral disc (IVD) degeneration is one of the leading causes of disability, and current therapies are mainly unsatisfactory. The key pathological feature during IVD degeneration is the dysfunction of annulus fibrosus (AF). Although tissue-engineered AF has shown great promise for IVD regeneration, the design and fabrication of biomimetic AF scaffold remains a challenge due to the complexity of its structure. Nowadays, 3D printing technology has drawn great attention due to its customizable processes and ability to produce complex tissue architecture. However, few existing 3D printing methods can accurately replicate the fine angle-ply architecture of native AF, which is one of the most critical steps for IVD regeneration, due to the limited printing resolution. In this study, we aimed to fabricate high-resolution polycaprolactone (PCL) scaffolds using a newly developed electrohydrodynamic 3D printing technique. The structural advantages of such scaffolds were verified by finite element analysis (FEA). The PCL scaffolds were further assembled into AF construct to replicate the angle-ply architecture of AF. The optimal assembling method was confirmed by FEA and mechanical tests. Thein vitroexperiments showed that the 3D printed AF scaffolds presented favorable biocompatibility and supported the adhesion and growth of AF cells. Thein vivoperformance of tissue-engineered IVDs (TE-IVDs), which consisted of 3D printed AF scaffold and GelMA hydrogel that simulated nucleus pulposus (NP), were evaluated using a rat total disc replacement model. We found that the implantation of TE-IVDs helped maintain the disc height, reduced the loss of NP water content, and partially restored the biomechanical function of IVD. In addition, the TE-IVDs achieved well integration with adjacent tissues and promoted new tissue formation. In summary, being able to accurately simulate the structural characteristics of native AF, the 3D printed angle-ply AF scaffolds hold potential for future applications in IVD regeneration.

The role of microenvironment in stem cell-based regeneration of intervertebral disc.

Regenerative medicine for intervertebral disc (IVD) disease, by utilizing chondrocytes, IVD cells, and stem cells, has progressed to clinical trials in the treatment of back pain, and has been studied in various animal models of disc degeneration in the past decade. Stem cells exist in their natural microenvironment, which provides vital dynamic physical and chemical signals for their survival, proliferation and function. Long-term survival, function and fate of mesenchymal stem cells (MSCs) depend on the microenvironment in which they are transplanted. However, the transplanted MSCs and the endogenous disc cells were influenced by the complicated microenvironment in the degenerating disc with the changes of biochemical and biophysical components. It is important to understand how the MSCs and endogenous disc cells survive and thrive in the harsh microenvironment of the degenerative disc. Furthermore, materials containing stem cells and their natural microenvironment have good clinical effects. However, the implantation of tissue engineering IVD (TE-IVD) cannot provide a complete and dynamic microenvironment for MSCs. IVD graft substitutes may need further improvement to provide the best engineered MSC microenvironment. Additionally, the IVD progenitor cells inside the stem cell niches have been regarded as popular graft cells for IVD regeneration. However, it is still unclear whether actual IVD progenitor cells exist in degenerative spinal conditions. Therefore, the purpose of this review is fourfold: to discuss the presence of endogenous stem cells; to review and summarize the effects of the microenvironment in biological characteristics of MSC, especially those from IVD; to explore the feasibility and prospects of IVD graft substitutes and to elaborate state of the art in the use of MSC transplantation for IVD degeneration in vivo as well as their clinical application.

Multifunctional Nanofibrous Scaffolds with Angle-Ply Microstructure and Co-Delivery Capacity Promote Partial Repair and Total Replacement of Intervertebral Disc.

There is an urgent clinical need for the treatment of annulus fibrosus (AF) impairment caused by intervertebral disc (IVD) degeneration or surgical injury. Although repairing injured AF through tissue engineering is promising, the approach is limited by the complicated angle-ply microstructure, inflammatory microenvironment, poor self-repairing ability of AF cells and deficient matrix production. In this study, electrospinning technology is used to construct aligned core-shell nanofibrous scaffolds loaded with transforming growth factor-ß3 (TGFß3) and ibuprofen (IBU), respectively. The results confirm that the rapid IBU release improves the inflammatory microenvironment, while sustained TGFß3 release enhances nascent extracellular matrix (ECM) formation. Biomaterials for clinical applications must repair local AF defects during herniectomy and enable AF regeneration during disc replacement, so a box defect model and total IVD replacement model in rat tail are constructed. The dual-drug delivering electrospun scaffolds are assembled into angle-ply structure to form a highly biomimetic AF that is implanted into the box defect or used to replace the disc. In two animal models, it is found that biomimetic scaffolds with good anti-inflammatory ability enhance ECM formation and maintain the mechanical properties of IVD. Findings from this study demonstrate that the multifunctional nanofibrous scaffolds provide inspirations for IVD repair.

Endothelialized microvessels fabricated by microfluidics facilitate osteogenic differentiation and promote bone repair.

In bone tissue engineering, vascularization is one of the critical factors that limit the effect of biomaterials for bone repair. While various approaches have been tried to build vascular networks in bone grafts, lack of endothelialization still constitutes a major technical hurdle. In this study, we have developed a facile technique to fabricate endothelialized biomimetic microvessels (BMVs) from alginate-collagen composite hydrogels within a single step using microfluidic technology. BMVs with different sizes could be readily prepared by adjusting the flow rate of microfluids. All BMVs supported perfusion and outward penetration of substances in the tube. Endothelial cells could adhere and proliferate on the inner wall of tubes. It was also found that the expression of CD31 and secretion of BMP-2 and PDGF-BB were higher in the rat umbilical vein endothelial cells (RUVECs) in BMVs than those cultured on hydrogel. When co-cultured with bone marrow mesenchymal stem cells (BMSCs), endothelialized BMVs promoted the osteogenic differentiation of BMSCs compared to those in acellular BMV group. In vivo, markedly enhanced new bone formation was achieved by endothelialized BMVs in a rat critical-sized calvarial defect model compared to those with non-endothelialized BMVs or without BMVs. Together, findings from both in vitro and in vivo studies have proven that endothelialized BMVs function to facilitate osteogenesis and promote bone regeneration, and therefore might present an effective strategy in bone tissue engineering. STATEMENT OF SIGNIFICANCE: In bone tissue engineering, limited vascularization is one of the critical factors that limit the effect of biomaterials for bone repair. In this study, we developed a facile technique to fabricate endothelialized biomimetic microvessels (BMVs) from alginate-collagen composite hydrogels within a single step using microfluidic technology. Both in vitro and in vivo studies have proven that endothelialized BMVs function to facilitate osteogenesis and promote bone regeneration, and therefore might present an effective strategy in bone tissue engineering.

CD271 antibody-functionalized microspheres capable of selective recruitment of reparative endogenous stem cells for in situ bone regeneration.

In the strategy of in situ bone regeneration, it used to be difficult to specifically recruit bone marrow mesenchymal stem cells (BM-MSCs) by a single marker. Recently, CD271 has been considered to be one of the most specific markers to isolate BM-MSCs; however, the effectiveness of CD271 antibodies in recruiting BM-MSCs has not been explored yet. In this study, we developed novel CD271 antibody-functionalized chitosan (CS) microspheres with the aid of polydopamine (PDA) coating to recruit endogenous BM-MSCs for in situ bone regeneration. The CS microspheres were sequentially modified with PDA and CD271 antibody through dopamine self-polymerization and bioconjugation, respectively. In vitro studies showed that the CD271 antibody-functionalized microspheres selectively captured significantly more BM-MSCs from a fluorescently labeled heterotypic cell population than non-functionalized controls. In addition, the PDA coating was critical for supporting stable adhesion and proliferation of the captured BM-MSCs. Effective early recruitment of CD271+ stem cells by the functionalized microspheres at bone defect site of SD rat was observed by the CD271/DAPI immunofluorescence staining, which led to significantly enhanced new bone formation in rat femoral condyle defect over long term. Together, findings from this study have demonstrated, for the first time, that the CD271 antibody-functionalized CS microspheres are promising for in situ bone regeneration.

Calcium phosphate bone cement with enhanced physicochemical properties via in situ formation of an interpenetrating network.

Calcium phosphate cement (CPC), which exhibits excellent biocompatibility and bioactivity, is a well-established material for the repair of bone defects. However, its disadvantages such as poor washout resistance and low mechanical strength limit its clinical applications. In this study, CPC with enhanced washout resistance and mechanical properties has been developed by the in situ crosslinking of glycidyl methacrylate modified γ-polyglutamic acid (m-PGA) within the cement matrix, forming an interpenetrating network. Compared with unmodified CPC, the final setting time of the composite cements was shortened and its washout resistance was significantly improved. In addition, the composite cements showed enhanced mechanical strength and degradation properties. An in vitro study demonstrated that the composite cements exhibited good biocompatibility. The in vivo results showed that the composite cements promoted bone formation. These results suggest that the biocompatible, injectable α-tricalcium phosphate (α-TCP)/m-PGA cements may have the potential to be used as bone filling materials for future clinical applications.

Mechano-regulation of vascular network formation without branches in 3D bioprinted cell-laden hydrogel constructs.

Restoration of a wound is a common surgical procedure in clinic. Currently, the skin required for clinical use is taken from the patient's own body. However, it can be difficult to obtain enough skin sources for large-sized wounds and thus surgeons have started using commercial skin substitutes. The current commercial skin, which includes epidermis substitute, dermis substitute, and bilateral skin substitute, has been popularized in clinic. However, the application is limited by the occurrence of ischemia necrosis after transplantation. Recent studies suggest the use of pre-vascularized skin substitutes for wound healing is a promising area in the research field of skin tissue engineering. Pre-vascularization can be induced by changes in cultivation periods, exertion of mechanical stimuli, or coculture with endothelial cells and various factors. However, few methods could control the formation of vascular branches in engineering tissue in a self-assembly way. In this study, we use three-dimensional (3D) printing technology to confirm that a mechanical force can control the growth of blood vessels in the direction of mechanical stimulation with no branches, and that Yes-associated protein activity is involved in the regulatory progress. In vivo experiments verified that the blood vessels successfully function for blood circulation, and maintain the same direction. Results provide a theoretical basis for products of pre-vascularized skin tissues and other organs created by 3D bioprinting.

Bladder muscle regeneration enhanced by sustainable delivery of heparin from bilayer scaffolds carrying stem cells in a rat bladder partial cystectomy model.

In bladder tissue engineering, regeneration of muscle is of equal importance to epithelial regeneration. However, as yet there is no effective strategy for promoting bladder muscle regeneration. In this study we aim to promote bladder muscle regeneration by sustainably delivering heparin from a bilayer scaffold carrying stem cells. The bilayer scaffold [heparin-polycaprolactone (PCL)/bladder decellularized matrix (BAM) Hep-PB/PCL] comprises an electrospun layer (Hep-PB electrospun membrane) and a three-dimensional (3D) printed layer (PCL scaffold), fabricated via coaxial-electrospinning and 3D printing, respectively. Heparin was encapsulated into the core of the Hep-PB fibers with a core-shell structure to sustain its release. The morphology of the bilayer scaffold and the microstructure of the electrospun fibers were characterized. The release behavior of heparin from various electrospun membranes was evaluated. The role of Hep-PB in promoting myogenic differentiation of the adipose-derived stem cells (ADSCs) through sustainable release of heparin was also evaluated. After 7 d culture, Hep-PB/PCL scaffolds carrying ADSCs (defined as ASHP) were used for bladder reconstruction in a rat partial cystotomy model. The result shows that the PCL printed scaffold has ordered macropores (∼370 µm), unlike the compact microstructure of electrospun films. The Hep-PB membrane exhibits a sustained release behavior for heparin. This membrane also shows better growth and proliferation of ADSCs than the other membranes. The polymerase chain reaction results show that the expression of smooth muscle cell markers in ADSCs is enhanced by the Hep-PB scaffold. The results of retrograde urethrography and histological staining indicate that the bladder volume in the ASHP group recovers better, and the regenerated bladder muscle bundles are arranged in a more orderly fashion compared with the direct suture and bladder decellularized matrix groups. Therefore, findings from this study show that bladder muscle regeneration could be enhanced by bilayer scaffolds delivering heparin and carrying stem cells, which may provide a new strategy for bladder tissue engineering.

Enhanced Osseointegration of Titanium Implants by Surface Modification with Silicon-doped Titania Nanotubes.

INTRODUCTION: Despite great progress made in developing orthopedic implants, the development of titanium (Ti) implants with ideal early osseointegration remains a big challenge. Our pilot study has demonstrated that Si-TiO2 nanotubes on the surface of Ti substrates could enhance their osteogenic activity. Hence, in this study, we aim to comprehensively evaluate the effects of silicon-doped titania (Si-TiO2) nanotubes on the osseointegration property of Ti implants. MATERIALS AND METHODS: The Ti implants were surface modified with Si-TiO2 nanotubes through in situ anodization and Si plasma immersion ion implantation (PIII) method. Three groups were divided as Ti implants (Ti), Ti modified with TiO2 nanotubes (TiO2-NTs) and Ti modified with Si-TiO2 nanotubes (Si-TiO2-NTs). The morphology of Si-TiO2 nanotubes was observed by scanning electron microscope. The growth and osteogenic differentiation of MC3T3-E1 cells on the Ti implants were evaluated. Further, the pull-out tests and in vivo osseointegration ability evaluation were performed after implanting the screws in the femur of Sprague Dawley rats. RESULTS: The Si-TiO2 nanotubes could be seen on the surface of Ti implants. The MC3T3-E1 cells could grow on the surface of Ti, TiO2-NTs and Si-TiO2-NTs, and showed fast proliferation rate on the Si-TiO2-NTs. Moreover, the production of some osteogenesis-related proteins (ALP and Runx2) at one week and calcium deposition at four week was also enhanced in Si-TiO2-NTs rather than other groups. In vivo osseointegration results showed that Si-TiO2 nanotube-modified Ti screws had higher pullout force at two and four weeks as well as enhanced new bone formation at six weeks compared to bare Ti screws and Ti screws modified with TiO2 nanotubes alone. DISCUSSION: The modification of Si-TiO2-NTs on the Ti substrate could generate a nanostructured and hydrophilic surface, which can promote cell growth. Moreover, the existence of the TiO2 nanotubes and Si element also can improve the in vitro osteogenic differentiation of MC3T3-E1 cells and early bone formation around the implanted screws. Together, findings from this study show that surface modification of Ti implants with Si-TiO2 nanotubes could enhance early osseointegration and therefore has the potential for clinical applications.