Biocompatibility In-vitro of Gel/HA Composite Scaffolds Containing Nano-Bioactive Glass for Tissue Engineering

Designing and fabricating nanocomposite scaffolds for bone regeneration from different biodegradable polymers and bioactive materials are an essential step to engineer tissues. In this study, the composite scaffold of gelatin/hyaluronic acid (Gel/HA) containing nano-bioactive glass (NBG) was prepared by using freeze-drying method. The biocompatibilities in-vitro of the Gel-HA/NBG composite scaffolds, including MTT assay, ALP activity, von Kossa staining and tetracycline staining, were investigated. The SEM observations revealed that the prepared scaffolds were porous with three-dimensional (3D) and interconnected microstructure, agglomerated NBG particles were uniformly dispersed in the matrix. MTT results indicated that the tested materials didn't show any cytotoxicity. The presence of NBG in the composite scaffold further enhanced the ALP activity in comparison with the pure Gel/HA scaffold. The von Kossa staining and tetracycline staining results also indicated that the NBG may improve the cell response. Therefore, the results indicated the nanocomposite scaffold made from Gel, HA and NBG particles could be considered as a potential bone tissue engineering implant.     

Fabrication and Properties of Porous Collagen/Hyaluronic Composite Scaffolds Containing Nano-Bioactive Glass for Tissue Engineering

In this work, nano-structured scaffolds were designed for tissue engineering using collagen, hyaluronic acid (HA) and nano-bioactive glass (NBAG) as their main components. The scaffold was prepared via freeze-drying method and the properties including morphology, porosity, compressive strength, swelling ratio and cytotoxicity in-vitro, were also evaluated. The composite scaffolds showed well interconnected macropores with the pore size of ranging from 100 to 500 μm. The porosity percent and swelling ability were decreased with the introduction of NBAG into the collagen/HA hydrogel; however, the compressive strength was enhanced. The cytotoxicity in-vitro study shows that the collagen-HA/NBAG scaffolds have good biocompatibility with improving effect on fibroblastic cells growth. It could be concluded that this scaffold fulfills the main requirements to be considered as a bone substitute.     

A comparative study of the in vitro degradation of poly(l-lactic acid)/β-tricalcium phosphate scaffold in static and dynamic simulated body fluid

Porous poly(l-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) composite is a new promising scaffold for bone tissue engineering. Porous scaffolds fabricated by liquid anti-solvent precipitation principle were subjected to degradation in dynamic simulated body fluid (DSBF) and in static simulated body fluid (SSBF) at 37 °C for 24 weeks, respectively. Results indicated that a large number of apatite layer were formed on the scaffolds. The results further indicated that SBF flow decreased the degradation rate of molecular weight and compressive strength significantly. The porosity and mass changes were related to the apatite formation and SBF flow. All the results might be owed to the mutual effects of the flow of SBF and the addition of β-TCP. The degradation rate of scaffolds could be adjusted by the additional fraction of β-TCP to meet the requirements of application in vivo.     

Development of porous,antibacterial and biocompatible GO/n-HAp/bacterial cellulose/β-glucan biocomposite scaffold for bone tissue engineering

Due to their potential renewable materials-based tissue engineering scaffolds has gained more attention. Therefore, researchers are looking for new materials to be used as a scaffold. In this study, we have focused on the development of a nanocomposite scaffold for bone tissue engineering (using bacterial cellulose (BC) and β-glucan (β-G)) via free radical polymerization and freeze-drying technique. Hydroxyapatite nanoparticles (n-HAp) and graphene oxide (GO) were added as reinforcement materials. The structural changes, surface morphology, porosity, and mechanical properties were investigated through spectroscopic and analytical techniques like Fourier transformation infrared (FT-IR), scanning electron microscope (SEM), Brunauer–Emmett-Teller (BET), and universal testing machine Instron. The scaffolds showed remarkable stability, aqueous degradation, spongy morphology, porosity, and mechanical properties. Antibacterial activities were performed against gram -ive and gram + ive bacterial strains. The BgC-1.4 scaffold was found more antibacterial compared to BgC-1.3, BgC-1.2, and BgC-1.1. The cell culture and cytotoxicity were evaluated using the MC3T3-E1 cell line. More cell growth was observed onto BgC-1.4 due to its uniform interrelated pores distribution, surface roughness, better mechanical properties, considerable biochemical affinity towards cell adhesion, proliferation, and biocompatibility. These nanocomposite scaffolds can be potential biomaterials for fractured bones in orthopedic tissue engineering.     

Porous nano-minerals substituted apatite/chitin/pectin nanocomposites scaffolds for bone tissue engineering

In the current study, a porous 3D scaffold using Gallium-Apatite/chitin/pectin (Ga-HA/C/P) nanocomposites scaffolds (NCS) were fabricated by freeze-drying process with applications in orthopedics (bone tissue engineering). Various NCSs (0%, 30%, 50 and 70%) were prepared and characterized for its chemical structure, crystalline phase, surface texture by using various techniques such as FT-IR, XRD and SEM-EDX, respectively. The analyses of physicochemical properties proved that the formulated scaffolds were highly porous, and mechanically stable with superior density. The nanocomposite scaffolds also presented with increased swelling ability, lower biodegradation rate and higher mechanical strength. Further, biocompatibility and cytotoxicity of Ga-HA/C/P nanocomposite scaffolds were studied using NIH3T3 cells and MG-63 cells revealed no toxicity and cells attached and proliferated on scaffolds. Further implantation of prepared NCS showed mature bone formation through formation of new bone cells and osteoblast differentiation. Also, Ga-HA/C/P nanocomposites scaffolds proved to be more effective than chitin-pectin composite scaffolds. Taking results together it can be inferred that the prepared nanocomposite scaffolds possesses the prerequisites and showed great potential for treating orthopedic applications.     

Synthesis and characterization of poly(d,l-lactide-co-glycolide) modified by maleic anhydride and 1,4-butanediamine

Bone tissue engineering is sought to apply strategies for bone defects healing biodegradable porous scaffolds without limitations and shortcomings. In this work, we have developed a novel maleic anhydride (MAH) and 1,4-butanediamine modified poly(lactide-co-glycolide) polymer (BMPLGA). The synthesized polymer was characterized by Fourier transform infrared spectrometry (FTIR), Nuclear magnetic resonance spectra (¹H NMR), gel permeation chromatography (GPC) and contact angle measurements. In addition, cell morphologies in the extracts and cell cytotoxity were also studied. The results showed that the BMPLGA was successfully obtained by introducing MAH and 1,4-butanediamine into PLGA in bulk. The introduction of anhydride and amino groups improved the hydrophilicity of PLGA. Fibroblastic cells showed normal morphologies in BMPLGA extracts, and the BMPLGA materials showed no cell cytotoxicity. The synthetic BMPLGA material may have potentials for biomedical applications due to improving hydrophilicity.     

Enhanced mechanical performance and biological evaluation of a PLGA coated β-TCP composite scaffold for load-bearing applications

Porous β-tricalcium phosphate (β-TCP) has been used for bone repair and replacement in clinics due to its excellent biocompatibility, osteoconductivity, and biodegradability. However, the application of β-TCP has been limited by its brittleness. Here, we demonstrated that an interconnected porous β-TCP scaffold infiltrated with a thin layer of poly(lactic-co-glycolic acid) (PLGA) polymer showed improved mechanical performance compared to an uncoated β-TCP scaffold while retaining its excellent interconnectivity and biocompatibility. The infiltration of PLGA significantly increased the compressive strength of β-TCP scaffolds from 2.90 to 4.19 MPa, bending strength from 1.46 to 2.41 MPa, and toughness from 0.17 to 1.44 MPa, while retaining an interconnected porous structure with a porosity of 80.65%. These remarkable improvements in the mechanical properties of PLGA-coated β-TCP scaffolds are due to the combination of the systematic coating of struts, interpenetrating structural characteristics, and crack bridging. The in vitro biological evaluation demonstrated that rat bone marrow stromal cells (rBMSCs) adhered well, proliferated, and expressed alkaline phosphatase (ALP) activity on both the PLGA-coated β-TCP and the β-TCP. These results suggest a new strategy for fabricating interconnected macroporous scaffolds with significantly enhanced mechanical strength for potential load-bearing bone tissue regeneration.     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

In vitro blood compatibility and bone mineralization aspects of polymeric scaffold laden with essential oil and metallic particles for bone tissue engineering

Bone tissue engineering scaffolds necessities appropriate physicochemical and mechanical properties to support its renewal. Electrospun scaffolds have been used unequivocally in bone tissue restoration. The main intention of this research is to develop electrospun polyurethane (PU) scaffold decorated with metallic particles and essential oil with advanced properties to make them as a putative candidate. The nanocomposite scaffold exhibited appropriate wettability and suitable fiber diameter compared to the polyurethane scaffold. Interaction of the added constituents with the polyurethane was corroborated through hydrogen bonding formation. Tensile strength of the composites was enhanced compared to the polyurethane scaffold. Thermal analysis depicted the lower weight loss of the composite scaffold than the pristine PU. Blood coagulation was significantly delayed and also the composite surface rendered safe interaction with red blood cells. In vitro toxicity testing using fibroblast cells portrayed the nontoxic behavior of the fabricated material. The above-said advanced properties of the composite scaffold can be warranted for bone tissue engineering application.     

Poly(l‐Lactic Acid)/Gelatin Fibrous Scaffold Loaded with Simvastatin/Beta‐Cyclodextrin‐Modified Hydroxyapatite Inclusion Complex for Bone Tissue Regeneration

Recently, the application of nanostructured materials in the field of tissue engineering has garnered attention to mediate treatment and regeneration of bone defects. In this study, poly(l ‐lactic acid) (PLLA)/gelatin (PG) fibrous scaffolds are fabricated and β‐cyclodextrin (βCD) grafted nano‐hydroxyapatite (HAp) is coated onto the fibrous scaffold surface via an interaction between βCD and adamantane. Simvastatin (SIM), which is known to promote osteoblast viability and differentiation, is loaded into the remaining βCD. The specimen morphologies are characterized by scanning electron microscopy. The release profile of SIM from the drug loaded scaffold is also evaluated. In vitro proliferation and osteogenic differentiation of human adipose derived stem cells on SIM/HAp coated PG composite scaffolds is characterized by alkaline phosphatase (ALP) activity, mineralization (Alizarin Red S staining), and real time Polymerase chain reaction (PCR). The scaffolds are then implanted into rabbit calvarial defects and analyzed by microcomputed tomography for bone formation after four and eight weeks. These results demonstrate that SIM loaded PLLA/gelatin/HAp‐(βCD) scaffolds promote significantly higher ALP activity, mineralization, osteogenic gene expression, and bone regeneration than control scaffolds. This suggests the potential application of this material toward bone tissue engineering.

     

Preparation of enzymatically cross-linked sulfated chitosan hydrogel and its potential application in thick tissue engineering

For the requirement of preliminary vascularization, the scaffolds for thick tissue engineering should possess not only good cell affinity, but also anticoagulant ability. In this paper, an enzymatically crosslinked hydrogel scaffold based on sulfated chitosan (SCTS) was prepared. Firstly, sulfated chitosan-hydroxyphenylpionic acid (SCTS-HPA) conjugate was synthesized, and its structure was identified by FITR and ¹H NMR. And then an enzymatically crosslinked hydrogel was prepared in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The gelation time, mechanical property, morphology and cytotoxicity to human umbilical vein endothelial cells (HUVECs) of the hydrogel was evaluated in vitro, the tissue compatibility of SCTS scaffold was studied in vivo. The results showed that the gelation time, mechanical property, morphology of the dehydrated hydrogel could be controlled by the HRP and H₂O₂ concentration. The cytotoxicity test showed that the hydrogel extracts had no cytotoxicity to HUVECs. The in vivo assay indicated that SCTS-HPA scaffold showed good tissue compatibility, and no thrombus formation. All these results indicated that the SCTS-HPA scaffold could be used as thick tissue engineering scaffold.     

In vitro degradation of porous poly(l-lactide-co-glycolide)/β-tricalcium phosphate (PLGA/β-TCP) scaffolds under dynamic and static conditions

In vitro degradation of porous poly(l-lactide-co-glycolide)/β-tricalcium phosphate (PLGA/β-TCP) scaffolds was studied by incubating the samples in phosphate buffered saline (PBS) at 37 °C and pH 7.4 under dynamic loading with respect to static conditions for 12 weeks. Under dynamic conditions, acidity of PBS was alleviated by the better solution circulation, and water absorption of the scaffolds increased more than that under static conditions in the first 8 weeks. Changes in mass, height, diameter, relative molecular mass and its distribution also happened more remarkably under dynamic conditions. Moreover, obvious cracks and a larger amount of β-TCP particles were observed on the wall of the scaffolds after degradation for 12 weeks under dynamic loading. Compressive modulus and strength showed an increase from the beginning to the 10th week but were lower after then. Results showed that degradation of PLGA/β-TCP scaffolds under dynamic conditions exhibited a significantly faster rate than that under static conditions.     

Production,blood compatibility and cytotoxicity evaluation of a single stage non-woven multicomponent electrospun scaffold mixed with sesame oil,honey and propolis for skin tissue engineering

Scaffolds used in skin tissue engineering must mimic the native function of the extracellular matrix (ECM) and facilitate the fibroblast cell response for new tissue growth. In this study, a novel dressing scaffold based on polyurethane (PU) with sesame oil, honey, and propolis was fabricated by electrospinning. Scanning electron microscopy (SEM) images showed that the diameter of the electrospun scaffolds decreased by blending sesame oil (784?±?125.46?nm) and sesame oil/honey/propolis (576?±?133.72?nm) into the PU matrix (890?±?116.911?nm). Fourier infrared (FT-IR) and thermogravimetric (TGA) analysis demonstrated the formation of hydrogen bonds and interaction between PU and sesame oil, honey, and propolis. Contact-angle measurement indicated reduced wettability of PU/sesame oil scaffold (114?±?1.732) and improved wettability (54.33?±?1.528) in the PU/sesame oil/honey/propolis scaffold. Further, tensile tests and atomic force microscopy (AFM) analysis indicated that the fabricated composite membrane exhibited enhanced mechanical strength and reduced surface roughness compared to the pristine PU. The developed composite displayed less toxicity to the red blood cells (RBC’s) compared to the pristine PU. Cytotoxicity assay showed enhanced cell viability of HDF in electrospun scaffolds than pristine PU after 72?h culture. These enhanced properties of the developed scaffolds suggest the potential of utilizing them in skin tissue engineering.     

Effect of a scaffold fabricated thermally from acetylated PLGA on the formation of engineered cartilage

A MHDS has been employed to fabricate 3D scaffolds from PLGA with acetyl endgroups to achieve in vivo regeneration of cartilage tissue. The fabricated acetylated-PLGA scaffold showed open pores and interconnected structures. Rabbit chondrocytes were seeded on the PLGA scaffolds and transplanted immediately into subcutaneous sites of athymic mice. Chondrocytes transplantation with untreated PLGA scaffolds served as a control. Histological analysis of the implants at 4 weeks with H&E staining and alcian blue staining revealed higher extracellular matrix and GAG expression at the neocartilage in the PLGA-6Ac scaffolds than that of the PLGA-6OH scaffold group. This endgroup-modified scaffold may be useful for successful cartilage tissue engineering in orthopedic applications.     

Synthesis and characterization of a nano‐hydroxyapatite/chitosan/polyethylene glycol nanocomposite for bone tissue engineering

A novel nanocomposite involving nano‐hydroxyapatite/chitosan/polyethylene glycol (n‐HAP/CS/PEG) has been successfully synthesized via co‐precipitation approach at room temperature. The purpose to synthesize such nanocomposite is to search for an ideal analogue which may mimick the composition of natural bone for bone tissue engineering with respect to suitable biocompatibility, cytotoxicity and mechanical properties. The FTIR spectra of n‐HAP/CS and n‐HAP/CS/PEG scaffolds indicated significant intermolecular interaction between the various components of both the nanocomposites. The results of XRD, TEM and TGA/DTA suggested that the crystallinity and thermal stability of the n‐HAP/CS/PEG scaffold have decreased and increased respectively, relative to n‐HAP/CS scaffold. The comparison of SEM images of both the scaffolds indicated that the incorporation of PEG influenced the surface morphology while a better in‐vitro bioactivity has been observed in n‐HAP/CS/PEG than in n‐HAP/CS based on SBF study, referring a greater possibility for making direct bond to living bone if implanted. Furthermore, MTT assay revealed superior non‐toxic nature of n‐HAP/CS/PEG to murine fibroblast L929 cells as compared to n‐HAP/CS. The comparative swelling studies of n‐HAP/CS/PEG and n‐HAP/CS scaffolds revealed a better swelling rate for n‐HAP/CS/PEG. Also n‐HAP/CS/PEG showed higher mechanical strength relative to n‐HAP/CS supportive of bone tissue ingrowths. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of surface-modified collagen on the adhesion, biocompatibility and differentiation of bone marrow stromal cells in poly(lactide-co-glycolide)/chitosan scaffolds

The material-driven differentiation of bone marrow stromal cells (BMSCs) is a critical issue in regeneration medicine. In this study, we showed the differentiation of BMSCs in 3-D scaffolds consisting of collagen, poly(lactide-co-glycolide) (PLGA) and chitosan. The results revealed that the collagen-grafted PLGA/chitosan scaffolds yielded little cytotoxicity to BMSCs. The scaffold containing type I collagen of 640μg/mL was about 1.2 times the cell adhesion efficiency of the corresponding unmodified scaffold. In addition, the modification of type I collagen with the density of 640μg/mL increased about 1.3 times the cell viability and 1.2 times the biodegradation, respectively. The differentiation of BMSCs in PLGA/chitosan scaffolds produced osteoblasts with mineral deposition on the substrate. Moreover, the surface collagen promoted the formation of mineralized tissue and reduced the amount of phenotypic BMSCs in the constructs. However, the induction with neuron growth factor (NGF) inhibited osteogenesis and guided the differentiation of BMSCs towards neurons in the constructs. Therefore, the combination of collagen-functionalized PLGA/chitosan scaffolds, NGF and BMSCs can be promising in neural tissue engineering.     

明胶溶液中沉淀法制备纯相β-磷酸三钙

本文以明胶、硝酸钙Ca(NO₃)₂和磷酸氢二铵(NH₄)₂HPO₄为前驱体,初始Ca/P为1.5,制备了纯相的β-磷酸三钙.红外谱图和X射线晶体衍射结果表明,溶液中直接沉淀得到的产物为缺钙磷灰石,该产物在明胶浓度≥0.22 %(质量分数)时热转化为纯相的β-磷酸三钙.通过晶体尺寸计算和比表面积测定,缺钙磷灰石的晶体大小随着明胶用量的增加而变小.透射电镜结果显示溶液中直接沉淀的缺钙磷灰石呈针状形貌,经过高温煅烧后,针状的缺钙磷灰石将相互融合形成葡萄状的β-磷酸三钙.差热/热重结果表明,明胶与生成的缺钙磷灰石形成了化学键合,这将有助于吸附较多的水分子,随后水分子与缺钙磷灰石发生化学反应生成羟基磷灰石,羟基磷灰石继续与沉淀中的偏磷酸钙反应生成β-磷酸三钙.本文还研究了明胶对纯相β-磷酸三钙的生成机理.     

Controllable synthesis and characterization of porous polyvinyl alcohol/hydroxyapatite nanocomposite scaffolds via an in situ colloidal technique

During the last decades, there have been several attempts to combine bioactive materials with biocompatible and biodegradable polymers to create nanocomposite scaffolds with excellent biocompatibility, bioactivity, biodegradability and mechanical properties. In this research, the nanocomposite scaffolds with compositions based on PVA and HAp nanoparticles were successfully prepared using colloidal HAp nanoparticles combined with freeze-drying technique for tissue engineering applications. In addition, the effect of the pH value of the reactive solution and different percentages of PVA and HAp on the synthesis of PVA/HAp nanocomposites were investigated. The SEM observations revealed that the prepared scaffolds were porous with three dimensional microstructures, and in vitro experiments with osteoblast cells indicated an appropriate penetration of the cells into the scaffold's pores, and also the continuous increase in cell aggregation on the scaffolds with increase in the incubation time demonstrated the ability of the scaffolds to support cell growth. According to the obtained results, the nanocomposite scaffolds could be considered as highly bioactive and potential bone tissue engineering implants.     

聚(丙交酯-co-乙交酯)/β-磷酸三钙复合物的电纺研究

对生物可吸收聚(丙交酯-co-乙交酯)(poly(lactide-co-glycolide),PLGA)与β-磷酸三钙(-βTCP)复合物体系进行了电纺.研究了PLGA的浓度,-βTCP与PLGA比例,加料速度,电压,喷头与接收体之间的距离等因素对电纺过程的影响,制备出纳米纤维膜,并用扫描电镜(SEM)等对纤维膜进行表征.结果表明,电纺溶液浓度越高,或者加料速度越快,纳米纤维的直径越粗.力学实验显示,复合物中-βTCP的含量增加使纳米纤维膜的拉伸强度和杨氏模量下降.     

Fabrication and characterization of chitosan-gelatin/single-walled carbon nanotubes electrospun composite scaffolds for cartilage tissue engineering applications

Cartilage is a connective tissue with a slow healing rate due to lack in blood circulation and slow metabolism. Designing tissue engineering scaffolds modified based on its specific features can assist its natural regeneration process. In this study, the chitosan-gelatin/single-walled carbon nanotubes functionalized by COOH (SWNTs-COOH) nanocomposite scaffolds were fabricated through electrospinning. The effect of each component and different duration of cross-linking were assessed in terms of morphology, porosity, chemical structure, thermal behavior, mechanical properties, wettability, biodegradability, and in vitro cell culture study. Adding SWNTs-COOH decreased fiber diameter, water contact angle and degradation rate while increased tensile strength, hydrophilicity, stability and cell viability, due to their high intrinsic electrical conductivity, and mechanical properties and the presence of COOH functional groups in its structure. All the sample presented a porosity percentage of more than 80%, which is essential for tissue engineering scaffolds. The presence SWNTs-COOH did not have any adverse effect on cytocompatibility. The optimal cross-linking time increased the stability of the scaffolds in PBS. It can be concluded that the chitosan-gelatin/1wt% SWNTs-COOH scaffold can be appropriate for cartilage tissue engineering applications.