Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.     

Enhancing angiogenesis in collagen matrices by covalent incorporation of VEGF

Since the survival of ingrowing cells in biomaterials for regenerative processes largely depends on the supply of nutrients and oxygen, angiogenesis plays an important role in the development of new materials for tissue engineering. In this study we investigated the possibility of enhancing the angiogenic properties of collagen matrices by covalent incorporation of the vascular endothelial growth factor (VEGF). In a previous paper we already reported the use of homo- and heterobifunctional cross-linking agents for modifying collagen matrices [1]. In the present work the angiogenic growth factor was linked to the collagen with the homobifunctional cross-linker disuccinimidyldisuccinatepolyethyleneglycol (SS-PEG-SS) in a two step procedure. The efficiency of the first reaction step–the reaction of SS-PEG-SS with VEGF—was evaluated by western blot analysis. After 10 minutes virtually all of the dimeric molecules VEGF were on average modified by conjugation with 1 cross-linking molecule. The biological activity of the conjugate was investigated by exposing endothelial cells to non-modified VEGF and to VEGF conjugated to the cross-linker. The conjugation only had a limited effect on the mitogenic activity of VEGF. We therefore applied the cross-linking reaction to the VEGF-collagen system. In a first approach the changes were evaluated by the in vitro exposure of HUVECs to non-modified matrices, to matrices in which the VEGF was simply admixed and to matrices in which the VEGF was covalently incorporated. The angiogenic properties were evaluated in vivo with the chorioallantois membrane model. In this assay the chorioallantois membrane of the chicken embryo was exposed to the same set of matrices. The covalent incorporation of VEGF has a small but significant effect both on the formation of microvessels in the chorioallantois membrane and the tissue ingrowth into the implant. The covalent incorporation of angiogenic growth factors may thus be considered as a promising approach for enhancing the angiogenic capabilities of collagen matrices. Also the cross-linking with the homobifunctional cross-linking agent has a positive effect on the angiogenic potential of the collagen matrices.     

Study of haemodialysis materials; physico-chemical and biological characterization of EVALVA,EVAPA and heparinized EVAPA

Partially hydrolyzed ethylene/vinyl acetate copolymers were modified by the covalent binding of a heparin-complexing polymer and further heparinized in order to improve their blood compatibility. These heparinizable polymeric materials (EVAPA) were obtained by a two-step reaction between an ethylene/vinyl alcohol/vinyl acetate (EVALVA) terpolymer, and the heparin complexing polymer N₂LL. The physico-chemical characterization of EVALVA, EVAPA and heparinized-EVAPA was carried out through thermal analysis, SEM, contact angle, potentiometric measurements, water uptake and FT-IR spectroscopic measurements. The biocompatibility of the above-mentioned samples was evaluated usingin vitro methods, through the determination of heparin release in phosphate buffer solution (PBS) and in human plasma, and with the investigation of hemostasis activation.     

Bioresorbable,heparinized polymers for stent coating:in vitro studies on heparinization efficiency,maintenance of anticoagulant properties and improvement of stent haemocompatibility

Biodegradable poly-(D,L-lactide) RESOMER^® R208 and poly(D,L-lactide-co-glycolide) RESOMER^® RG756 were heparinized to improve the blood contacting properties of the materials. The immobilization of heparin was performed with glutaraldehyde as coupling agent. The efficacy of the surface modification was monitored with respect to the total amount of bound heparin measured by a toluidine blue assay, the anticoagulant potential estimated by a factor Xa assay, and the activation of platelets estimated by a GMP140 assay. It was found that a reaction at ambient temperature for 2h resulted in optimal heparin binding, with high anticoagulant activity and low thrombogenicity. The storage of heparinized polylactide in saline solutions up to 8 days demonstrated the release of small quantities of heparin into the fluid. A further finding was that with prolonged storage the anticoagulant potential was improved, whereas the thrombogenicity decreased. Comparison of platelet activation on RESOMER^® R208 as unmodified and heparinized material with polypropylene and Pellethane^® revealed that heparinization of R208 substantially improved the haemocompatibility. Coating and subsequent heparinization of intravascular stents were carried out with RESOMER^® RG756 because of its more appropriate mechanical properties.In vitro studies with blood under flow conditions demonstrated that platelet activation on Palmaz stents was considerably diminished after polymer coating and heparinization.This paper was accepted for publication after the 1995 Conference of the European Society of Biomaterials, Oporto, Portugal, 10–13 September.     

Functional Nanocomplexes with Vascular Endothelial Growth Factor A/C Isoforms Improve Collateral Circulation and Cardiac Function

Protein‐based therapies are potential treatments for cancer, immunological, and cardiovascular diseases. However, effective delivery systems are needed because of their instability, immunogenicity, and so on. Crosslinked negatively charged heparin polysaccharide nanoparticle (HepNP) is proposed for protein delivery. HepNP can efficiently condense vascular endothelial growth factor (VEGF) because of the unique electronegative sulfonic acid and carboxyl domain of heparin. HepNP is then assembled with VEGF‐C (Hep@VEGF‐C) or VEGF‐A (Hep@VEGF‐A) protein for the therapy of myocardial infarction (MI) via intravenous (iv) injection. Hep@VEGF‐A‐mediated improvement of cardiac function by promoting angiogenesis is limited because of elevated vascular permeability, while Hep@VEGF‐C effectively promotes lymphangiogenesis and reduces edema. On this basis, a graded delivery of VEGF‐C (0.5–1 h post‐MI) and VEGF‐A (5 d post‐MI) using HepNP is developed. At the dose ratio of 3:1 (Hep@VEGF‐C vs Hep@VEGF‐A), Hep@VEGF functional complexes substantially reduce the scar formation (≈?39%; p < 0.05) and improve cardiac function (≈+74%; p < 0.05). Such a HepNP delivery system provides a simple and effective therapeutic strategy for cardiovascular diseases by delivering functional proteins. Because of the unique binding ability of heparin with cytokines and growth factors, HepNP also has considerable application prospects in protein therapy for other serious diseases.     

Emulsion electrospun vascular endothelial growth factor encapsulated poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactic acid-<Emphasis Type="Italic">co</Emphasis>-ε-caprolactone) nanofibers for sustained release in cardiac tissue engineering

Emulsion electrospinning is a novel approach to fabricate core–shell nanofibers, and it is associated with several advantages such as the alleviation of initial burst release of drugs and it protects the bioactivity of incorporated drugs or proteins. Aiming to develop a sustained release scaffold which could be a promising substrate for cardiovascular tissue regeneration, we encapsulated vascular endothelial growth factor (VEGF) with either of the protective agents, dextran or bovine serum albumin (BSA) into the core of poly(l-lactic acid-co-ε-caprolactone) (PLCL) nanofibers by emulsion electrospinning. The morphologies and fiber diameters of the emulsion electrospun scaffolds were determined by scanning electron microscope, and the core–shell structure was evaluated by laser scanning confocal microscope. Uniform nanofibers of PLCL, PLCL–VEGF–BSA, and PLCL–VEGF–DEX with fiber diameters in the range of 572 ± 92, 460 ± 63, and 412 ± 61 nm, respectively were obtained by emulsion spinning. The release profile of VEGF in phosphate-buffered saline for up to 672 h (28 days) was evaluated, and the scaffold functionality was established by performing cell proliferations using human bone marrow derived mesenchymal stem cells. Results of our study demonstrated that the emulsion electrospun VEGF containing core–shell structured PLCL nanofibers offered controlled release of VEGF through the emulsion electrospun core–shell structured nanofibers and could be potential substrates for cardiac tissue regeneration.     

Substrate stiffness affects skeletal myoblast differentiation in vitro

Abstract

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.     

Acceleration of segmental bone regeneration in a rabbit model by strontium-doped calcium polyphosphate scaffold through stimulating VEGF and bFGF secretion from osteoblasts

The development of suitable bioactive three-dimensional scaffold for the promotion of bone regeneration is critical in bone tissue engineering. The purpose of this study was to investigate in vivo osteogenesis of the porous strontium-doped calcium polyphosphate (SCPP) scaffolds for bone repair, as well as the relationship between osteogenic properties of SCPP scaffolds and the secretion of bFGF and VEGF from osteoblasts stimulated by SCPP. Besides, the advantages of scaffolds seeded with mesenchymal stem cells (MSCs) for bone repair were also studied. Firstly, the bone repair evaluation of scaffolds was performed on a rabbit segmental bony defects model over a period of 16 weeks by histology combined with X-ray microradiography. And then, in order to avoid the influence from the other factors such as hypoxia which emerge in vivo study and affect the secretion of VEGF and bFGF from host cells, human osteoblast-like cells (MG63) were seeded to SCPP, CPP and HA scaffolds in vitro to determine the ability of these scaffolds to stimulate the secretion of angiogenic growth factors (VEGF and bFGF) from MG63 and further explore the reason for the better osteogenic properties of SCPP scaffolds. The histological and X-ray microradiographic results showed that the SCPP scaffolds presented better osteogenic potential than CPP and HA scaffolds, when combined with MSCs, the SCPP scaffolds could further accelerate the bone repair. And the amounts of VEGF measured by ELISA assay in SCPP, CPP and HA groups after cultured for 7 days were about 364.989 pg/mL, 244.035 pg/mL and 232.785 pg/mL, respectively. Accordingly, the amounts of bFGF were about 27.085 pg/mL, 15.727 pg/mL and 8.326 pg/mL. The results revealed that the SCPP scaffolds significantly enhanced the bFGF and VEGF secretion compared with other scaffolds. The results presented in vivo and in vitro study demonstrated that the SCPP could accelerate bone formation through stimulating the secretion of VEGF and bFGF from osteoblasts, making it attractive for bone regeneration.     

Angiogenic potential of boron-containing bioactive glasses: in vitro study

Boron-containing bioactive glasses (BGs) are being extensively researched for the treatment and regeneration of bone defects because of their osteostimulatory and neovascularization potential. In this study, we report the effects of the ionic dissolution products (IDPs) of different boron-doped, borosilicate, and borate BG scaffolds on mouse bone marrow stromal cells in vitro, using an angiogenesis assay. Five different BG scaffolds of the system SiO₂–Na₂O–K₂O–MgO–CaO–P₂O₅–B₂O₃ (with varying amounts of SiO₂ and B₂O₃) were fabricated by the foam replication technique. Bone marrow stromal cells were cultivated in contact with the IDPs of the boron-containing BG scaffolds at different concentrations for 48 h. The expression and secretion of vascular endothelial growth factor (VEGF) from the cultured cells was measured quantitatively using the VEGF ELISA Kit. Cell viability and cell morphology were determined using WST-8 assay and H&E staining, respectively. The cellular response was found to be dependent on boron content and the B release profile from the glasses corresponded to the positive or negative biological activity of the BGs.     

Modified Fibrin Hydrogels stimulate Angiogenesis in vivo: potential Application to increase Perfusion of Ischemic Tissues

The lack of angiogenesis in ischemic tissues is a major health problem and many studies aim to explore strategies to locally increase blood perfusion. Our approach is to use covalently modified fibrin‐based hydrogels as a matrix that induces endothelial cell survival in vitro and angiogenesis in vivo. Fibrin hydrogels were covalently modified by L1Ig6, a specific receptor for cell survival integrin αvβ3 that is expressed on angiogenic endothelial cells. In addition, L1Ig6‐modified matrices were filled with growth factors VEGF‐A₁₆₅ or bFGF. These hydrogels were applied on growing shell‐free chicken chorioallantoic membranes (CAMs) and the developing vasculature was found to be increased by ∼50 %. Moreover, the increase in αv‐integrin levels in the CAMs underlying the hydrogel implants were investigated and found to be increased by ∼40 % and ∼100 %, respectively, after CAM stimulation with L1Ig6 alone or in combination with growth factors VEGF‐A₁₆₅ and bFGF. Therefore, modified fibrin hydrogels provide an interesting way to design an implant that can be introduced at the site of ischemia, and provides a scaffold and release system for growth factors that induce specific tissue responses.     

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Abstract

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na⁺/K⁺-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.     

Sterilization of heparinized Cuprophan hemodialysis membranes

The effects of sterilization of dry heparinized Cuprophan hemodialysis membranes by means of ethylene oxide (EtO) exposure, gamma irradiation, or steam on the anticoagulant activity and chemical characteristics of immobilized heparin and the permeability of the membrane were investigated. Sterilization did not result in a release of heparin or heparin fragments from heparinized Cuprophan. Sterilization of heparinized Cuprophan by means of EtO exposure and gamma irradiation induced a slight, insignificant decrease of the anticoagulant activity. In contrast, steam-sterilized heparinized Cuprophan showed a higher anticoagulant activity than unsterilized heparinized Cuprophan, which was most likely caused by cleavage of some of the covalent bonds between heparin and Cupropha. The effects of sterilization on the permeability of unmodified Cuprophan and heparinized Cuprophan were compared. The permeability of unmodified Cuprophan for vitamin B₁₂ (Vit B₁₂) and sulfobromophthalein (SBP) was reduced by 20–35% after EtO exposure and gamma irradiation and was reduced by 90–95% after steam sterilization. The water permeability of unmodified Cuprophan remained the same after EtO exposure and gamma irradiation but also dramatically reduced after steam sterilization. These reductions were ascribed to the collapse of pores of the membrane. The permeability of heparinized Cuprophan was not affected by EtO exposure and gamma irradiation but dramatically reduced after steam sterilization, although to a lesser extent than in the case of unmodified Cuprophan. Apparently, the presence of immobilized heparin (partially) prevented the collapse of pores during sterilization. Gamma irradiation was recommended as the preferred method of sterilization for heparinized Cuprophan.     

Restoration of mandibular bone defects with demineralized bone matrix combined with three-dimensional cultured bone marrow-derived mesenchymal stem cells in minipig models

Mandibular defects, caused by congenital, pathological or iatrogenic insults, can significantly affect patient quality of life. The reconstruction of mandible has recently gained the interest of clinical and tissue engineering researchers. The purpose of this study was to evaluate the effectiveness of three-dimensional (3-D) cultured autologous grafts prepared using bone marrow-derived mesenchymal stem cells (BMSCs) combined with demineralized bone matrix (DBM) scaffolds for the restoration of mandibular defects. Cylindrical defects were created in the mandibular body of minipigs and filled with 3D-cultured BMSCs/DBM autografts, 2D-cultured BMSCs/DBM autografts, DBM material (without cells), or were left unfilled (blank). Using computed tomographic (CT) imaging and histological staining, we found that treatment of mandibular defects using 3-D cultured BMSCs/DBM autografts offered improvements in bone formation over both 2-D cultured autografts and cell-free DBM scaffolds. We found increased osteoid formation in 3D and 2D cultures, with more osteogenic cells present in the 3D constructs. We suggest that 3-D cultured homograft BMSCs combined with DBM scaffolds represents a new strategy for bone reconstruction, with potential future clinical applicability.     

Enhanced physicochemical properties of collagen by using EDC/NHS-crosslinking

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Crosslinking density, matrix morphology, swelling ratio shrinkage temperature and resistance against collagenase digestion were determined to evaluate the physicochemical properties of the collagen matrices with and without crosslinking. The results conformed that the porous structure of collagen was largely preserved and adjusted by crosslinking treatment. Furthermore, crosslinked collagen samples showed significantly reduced swelling ratio and increased resistance against thermal treatment and enzymatic degradation compared to non-crosslinked samples. An in vitro evaluation of MC3T3-E1 cells seeded onto the crosslinked and non-crosslinked collagen matrix indicated that crosslinked collagen was nontoxic and improved cell proliferation. Through this work, it was shown that an osteoconductive collagen matrix with optimized properties used as bioactive and bioresorbable scaffolds in bone tissue engineering could be fabricated through the EDC/NHS-crosslinking method.     

Stem cell differentiation on electrospun nanofibrous substrates for vascular tissue engineering

Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and requirements in vascular tissue regeneration. In our study, poly-l-lactide (PLLA) and hybrid PLLA/collagen (PLLA/Coll) nanofibers (3:1 and 1:1) with fiber diameters of 210 to 430 nm were fabricated by electrospinning. Their morphological, chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR), and tensile instrument, respectively. Bone marrow derived mesenchymal stem cells (MSCs) seeded on electrospun nanofibers that are capable of differentiating into vascular cells have great potential for repair of the vascular system. We investigated the potential of MSCs for vascular cell differentiation in vitro on electrospun PLLA/Coll nanofibrous scaffolds using endothelial differentiation media. After 20 days of culture, MSC proliferation on PLLA/Coll(1:1) scaffolds was found 256% higher than the cell proliferation on PLLA scaffolds. SEM images showed that the MSC differentiated endothelial cells on PLLA/Coll scaffolds showed cobblestone morphology in comparison to the fibroblastic type of undifferentiated MSCs. The functionality of the cells in the presence of ‘endothelial induction media’, was further demonstrated from the immunocytochemical analysis, where the MSCs on PLLA/Coll (1:1) scaffolds differentiated to endothelial cells and expressed the endothelial cell specific proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and Von Willebrand factor (vWF). From the results of the SEM analysis and protein expression studies, we concluded that the electrospun PLLA/Coll nanofibers could mimic the native vascular ECM environment and might be promising substrates for potential application towards vascular regeneration.     

Porous-conductive chitosan scaffolds for tissue engineering II. <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> degradation

Porous-conductive chitosan scaffolds were fabricated by blending conductive polypyrrole (PPy) particles with chitosan solution and employing an improved phase separation method. In vitro and in vivo degradation behaviors of these scaffolds were investigated. In the case of in vitro degradation, an enzymatic degradation system was employed and lysozyme was used as a working enzyme. Meanwhile, the degradation products of scaffolds, glucosamine and N-acetyl-glucosamine, were also analyzed with a HPLC method. In vivo degradation of scaffolds was performed by subcutaneously implanting these scaffolds in rat for prescheduled time intervals. In the both cases, the weight-loss of scaffolds was monitored during the whole degradation process for evaluating the degradation of scaffolds. The changes in conductivity of scaffolds afterin vitro or in vivo degradation were also measured using a four-point technique. It was observed that the pore parameters of scaffolds themselves could significantly influence the degradation behaviors of scaffolds but the PPy content in the scaffolds seemed not to impart its effect to the degradation of scaffolds. Degradation dynamics of scaffolds and conductivity measurements indicated that these scaffolds shown fairly different behaviors in their in vitro and in vivo degradation process. According to the results obtained from in vitro and in vivo degradation of scaffolds and based on some requirements of practical tissue engineering application, it was suggested that the PPy content in the scaffold should be slightly higher than 3 wt.% but lower than 6 wt.%.     

Evaluation of bone matrix and demineralized bone matrix incorporated PLGA matrices for bone repair

The aim of this study was to evaluate the composite matrices prepared using Poly(lactic-co-glycolic acid)- PLGA (85:15) by incorporating human bone matrix (BM) powder or demineralized bone matrix (DBM) powder with the weight ratio of polymer: BM or DBM (75:25) to apply for bone repair. Murine Bone Marrow Stromal Cell (BMSC) attachment was studied with different time points at 30 min, 1 h, 2 h, 4 h, and 6 h for BM/PLGA, DBM/PLGA and PLGA control matrices. All types of matrices were linearly increased the BMSC attachment with the increase of time. Significantly higher number of BMSCs was attached to the both BM/PLGA and DBM/PLGA matrices after 2 h compared to the controls. If BM or DBM is incorporated into biodegradable PLGA matrices and cultured with BMSCs, those composite matrices could be potentially used for bone tissue engineering applications. In addition, particle migration and handling difficulties in DBM powder in clinical applications eliminate using a PLGA matrix. Furthermore, we have observed that DBM/PLGA matrices were structurally stronger compared to the BM/PLGA or control PLGA matrices when they exposed to physiological environment for 72 days.     

Fabrication of a reticular poly(lactide-co-glycolide) cylindrical scaffold for the in vitro development of microvascular networks

Abstract

The microvascular network is a simple but critical system that is responsible for a range of important biological mechanisms in the bodies of all animals. The ability to generate a functional microvessel not only makes it possible to engineer vital tissue of considerable size but also serves as a platform for biomedical studies. However, most of the current methods for generating microvessel networks in vitro use rectangular channels which cannot represent real vessels in vivo and have dead zones at their corners, hence hindering the circulation of culture medium. We propose a scaffold-wrapping method which enables fabrication of a customized microvascular network in vitro in a more biomimetic way. By integrating microelectromechanical techniques with thermal reflow, we designed and fabricated a microscale hemi-cylindrical photoresist template. A replica mold of polydimethylsiloxane, produced by casting, was then used to generate cylindrical scaffolds with biodegradable poly(lactide-co-glycolide) (PLGA). Human umbilical vein endothelial cells were seeded on both sides of the PLGA scaffold and cultured using a traditional approach. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin on the cultured cell demonstrated the potential of generating a microvascular network with a degradable cylindrical scaffold. Our method allows cells to be cultured on a scaffold using a conventional culture approach and monitors cell conditions continuously. We hope our cell-covered scaffold can serve as a framework for building large tissues or can be used as the core of a vascular chip for in vitro circulation studies.     

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.     

Structure and photochromism of polyoxometalates nanoparticles in cross-linked polymer networks

Cross-linked polymer (P(VP-MB)) based on N-vinylpyrrolidone (VP) and N, N′-methylene-bis-acrylamide (MB) was used as polymer matrix to construct photochromic nanocomposite thin films with entrapping the Keggin-type polyoxometalates (POM). The microstructure, photochromic behavior, and mechanism of the films were studied with transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectra (UV-vis), and electron resonance spectra (ESR). The transparent films changed from colorless to blue under UV irradiation. The films showed good reversible photochromism and could recover the colorless state gradually in the air, where oxygen plays an important role during the bleaching process. The heteropolyanions dispersed in composite films uniformly and exhibited strong coulombic interaction with cross-linked polymer. Composite films contained molybdenum had higher photochromic efficiency and slower bleaching reaction than films contained tungsten. According to ESR, the photochromic mechanism was followed by charge-transfer happened between POM and cross-linked polymer.