Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering

We describe a comparative assessment of the structure–property–process relationship of three-dimensional chitosan–nanohydroxyapatite (nHA) and pure chitosan scaffolds in conjunction with their respective biological response with the aim of advancing our insight into aspects that concern bone tissue engineering. High- and medium-molecular-weight (MW) chitosan scaffolds with 0.5, 1 and 2 wt.% fraction of nHA were fabricated by freezing and lyophilization. The nanocomposites were characterized by a highly porous structure and the pore size (～50 to 120 μm) was in a similar range for the scaffolds with different content of nHA. A combination of X-ray diffraction, Fourier transform infrared spectroscopy and electron microscopy indicated that nHA particles were uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and nHA. The compression modulus of hydrated chitosan scaffolds was increased on the addition of 1 wt.% nHA from 6.0 to 9.2 kPa in high-MW scaffold. The water uptake ability of composites decreased with an increase in the amount of nHA, while the water retention ability was similar to pure chitosan scaffold. After 28 days in physiological condition, nanocomposites indicated about 10% lower degree of degradation in comparison to chitosan scaffold. The biological response of pre-osteoblasts (MC 3T3-E1) on nanocomposite scaffolds was superior in terms of improved cell attachment, higher proliferation, and well-spread morphology in relation to chitosan scaffold. In composite scaffolds, cell proliferation was about 1.5 times greater than pure chitosan after 7 days of culture and beyond, as implied by qualitative analysis via fluorescence microscopy and quantitative study through MTT assay. The observations related to well-developed structure morphology, physicochemical properties and superior cytocompatibility suggest that chitosan–nHA porous scaffolds are potential candidate materials for bone regeneration although it is necessary to further enhance the mechanical properties of the nanocomposite.     

Tetronic®-based composite hydrogel scaffolds seeded with rat bladder smooth muscle cells for urinary bladder tissue engineering applications

Natural hydrogels such as collagen offer desirable properties for tissue engineering, including cell adhesion sites, but their low mechanical strength is not suitable for bladder tissue regeneration. In contrast, synthetic hydrogels such as poly (ethylene glycol) allow tuning of mechanical properties, but do not elicit protein adsorption or cell adhesion. For this reason, we explored the use of composite hydrogel blends composed of Tetronic (BASF) 1107-acrylate (T1107A) in combination with extracellular matrix moieties collagen and hyaluronic acid seeded with bladder smooth muscle cells (BSMC). This composite hydrogel supported BSMC growth and distribution throughout the construct. When compared to the control (acellular) hydrogels, mechanical properties (peak stress, peak strain, and elastic modulus) of the cellular hydrogels were significantly greater. When compared to the 7-day time point after BSMC seeding, results of mechanical testing at the 14-day time point indicated a significant increase in both ultimate tensile stress (4.1–11.6 kPa) and elastic modulus (11.8–42.7 kPa) in cellular hydrogels. The time-dependent improvement in stiffness and strength of the cellular constructs can be attributed to the continuous collagen deposition and reconstruction by BSMC seeded in the matrix. The composite hydrogel provided a biocompatible scaffold for BSMC to thrive and strengthen the matrix; further, this trend could lead to strengthening the construct to match the mechanical properties of the bladder.     

Fabrication and characterization of sol-gel derived 45S5 Bioglass®-ceramic scaffolds

Although Bioglass® has existed for nearly half a century its ability to trigger bone formation and tuneable degradability is vastly superior to other bioceramics, such as SiO₂–CaO bioactive glasses. The sol–gel process of producing glass foams is well established for SiO₂–CaO compositions, but not yet established for 45S5 composites containing Na₂O. In this work the sol–gel derived 45S5 Bioglass® has for the first time been foamed into highly porous three-dimensional scaffolds using a surfactant, combined with vigorous mechanical stirring and subsequent sintering at 1000 °C for 2 h. It was found that the mechanical strength of the sintered sol–gel derived Bioglass® scaffolds was significantly improved, attributable to the small fraction of material on the pore walls. More importantly, the compressive strength of the three-dimensional scaffolds produced by this surfactant foaming method could be predicted using Gibson and Ashby’s closed cell model of porous networks. A comparative experiment revealed that ion release from the sol–gel derived Bioglass® foams was faster than that of counterparts produced by the replication technique. In vitro evaluation using osteoblast-like cells demonstrated that the sol–gel derived 45S5 Bioglass foams supported the proliferation of viable cell populations on the surface of the scaffolds, although few cells were observed to migrate into the virtually closed pores within the foams. Further work should be focused on modifications of the reaction conditions or alternative foaming techniques to improve pore interconnection.     

In vitro evaluation of 45S5 Bioglass®-derived glass-ceramic scaffolds coated with carbon nanotubes

Highly porous (> 90% porosity) 45S5 Bioglass?-derived glass-ceramic scaffolds were fabricated by foam replication method, and coated with carbon nanotubes (CNT) (coating thickness: 1 μm) using electrophoretic deposition (EPD). In vitro cell culture using mesenchymal stem cells (MSCs) was carried out on both scaffold systems (with and without CNT coating) over a 4-week period. By using AlamarBlue?, BSA and alkaline phosphatase assays; the cell viability and differentiation were measured quantitatively measured and compared between the two scaffold types. The results showed that both scaffold systems are biocompatible with MSCs and they can support the cellular activity. No cytotoxic effects of CNT were observed under the conditions of the present experiments. Although a lower initial cell viability on the CNT-coated scaffolds was observed, no significant differences were found after 4 weeks of culture compared with the uncoated scaffolds. This work therefore shows that there is in principle no significant improvement of cellular responses by creating a CNT-coating on this type of highly bioactive scaffolds. However, the electrical conductivity introduced by the coating might have the potential to increase cell viability and differentiation when cell culture is carried out under the effect of electrical stimulation.     

Preparation and characterization of novel β-chitin/nanodiopside/nanohydroxyapatite composite scaffolds for tissue engineering applications

β-chitin/nanodiopside/nanohydroxyapatite (CT/nDP/nHAp) composite scaffolds were synthesized from the combination of chitin, nDP, and nHAp in different inorganic/organic weight ratios by the freeze-drying technique. The prepared nHAp, and composite scaffolds were characterized by BET, TG, FT-IR, SEM, and XRD studies. The composite scaffolds had 50–75% porosities with well-defined interconnected porous networks. Moreover, investigation of the cell attachment and viability using MTT, DMEM solution, and mouse preosteoblast cell proved the cytocompatibile nature of the composite scaffolds with improved cell adhesion. All these results mainly illustrated that this composite could be a candidate for bone tissue engineering application.     

Balancing mechanical strength with bioactivity in chitosan–calcium phosphate 3D microsphere scaffolds for bone tissue engineering: air- vs. freeze-drying processes

The objective of this study was to evaluate the potential benefit of 3D composite scaffolds composed of chitosan and calcium phosphate for bone tissue engineering. Additionally, incorporation of mechanically weak lyophilized microspheres within those air-dried (AD) was considered for enhanced bioactivity. AD microsphere, alone, and air- and freeze-dried microsphere (FDAD) 3D scaffolds were evaluated in vitro using a 28-day osteogenic culture model with the Saos-2 cell line. Mechanical testing, quantitative microscopy, and lysozyme-driven enzymatic degradation of the scaffolds were also studied. FDAD scaffold showed a higher concentration (p?<?0.01) in cells per scaffold mass vs. AD constructs. Collagen was ～31% greater (p?<?0.01) on FDAD compared to AD scaffolds not evident in microscopy of microsphere surfaces. Alternatively, AD scaffolds demonstrated a superior threefold increase in compressive strength over FDAD (12 vs. 4?MPa) with minimal degradation. Inclusion of FD spheres within the FDAD scaffolds allowed increased cellular activity through improved seeding, proliferation, and extracellular matrix production (as collagen), although mechanical strength was sacrificed through introduction of the less stiff, porous FD spheres.     

Chitosan–poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: In vitro degradation and in vivo bone regeneration studies

Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation.     

Fabrication and characterization of poly(γ-glutamic acid)-graft-chondroitin sulfate/polycaprolactone porous scaffolds for cartilage tissue engineering

The development of blended biomacromolecule and polyester scaffolds can potentially be used in many tissue engineering applications. This study was to develop a poly(γ-glutamic acid)-graft-chondroitin sulfate-blend-poly(ε-caprolactone) (γ-PGA-g-CS/PCL) composite biomaterial as a scaffold for cartilage tissue engineering. Chondroitin sulfate (CS) was grafted to γ-PGA, forming a γ-PGA-g-CS copolymer with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) system. The γ-PGA-g-CS copolymers were then blended with PCL to yield a porous γ-PGA-g-CS/PCL scaffold by salt leaching. These blended scaffolds were characterized by ¹H NMR, ESCA, water-binding capacity, mechanical test, degradation rate and CS assay. The results showed that with γ-PGA-g-CS as a component, the water-binding capacity and the degradation rate of the scaffolds would substantially increase. During a 4 week period of culture, the mechanical stability of γ-PGA-g-CS/PCL scaffolds was raised gradually and chondrocytes were induced to function normally in vitro. Furthermore, a larger amount of secreted GAGs was present in the γ-PGA-g-CS/PCL matrices than in the control (PCL), as revealed by Alcian blue staining of the histochemical sections. Thus, γ-PGA-g-CS/PCL matrices exhibit excellent biodegradation and biocompatibility for chondrocytes and have potential in tissue engineering as temporary substitutes for articular cartilage regeneration.     

Fabrication and characterization of chitosan/OGP coated porous poly(ε-caprolactone) scaffold for bone tissue engineering

As one of the stimulators on bone formation, osteogenic growth peptide (OGP) improves both proliferation and differentiation of the bone cells in vitro and in vivo. The aim of this work was the preparation of three dimensional porous poly(ε-caprolactone) (PCL) scaffold with high porosity, well interpore connectivity, and then its surface was modified by using chitosan (CS)/OGP coating for application in bone regeneration. In present study, the properties of porous PCL and CS/OGP coated PCL scaffold, including the microstructure, water absorption, porosity, hydrophilicity, mechanical properties, and biocompatibility in vitro were investigated. Results showed that the PCL and CS/OGP-PCL scaffold with an interconnected network structure have a porosity of more than 91.5, 80.8%, respectively. The CS/OGP-PCL scaffold exhibited better hydrophilicity and mechanical properties than that of uncoated PCL scaffold. Moreover, the results of cell culture test showed that CS/OGP coating could stimulate the proliferation and growth of osteoblast cells on CS/OGP-PCL scaffold. These finding suggested that the surface modification could be a effective method on enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering application and the developed porous CS/OGP-PCL scaffold should be considered as alternative biomaterials for bone regeneration.     

A novel collagen/hydroxyapatite/poly(lactide-co-ε-caprolactone) biodegradable and bioactive 3D porous scaffold for bone regeneration

The goal of this study was to design a nontoxic scaffold with both composition and microstructure suitable for bone engineering using collagen (Coll), hydroxyapatite (HA), and poly(lactide-co-ε-caprolactone) (PLCL). Mineralized type I Coll was produced by direct nucleation of HA particles inside self-assembled Coll fibers to obtain a Coll/HA complex, which was then added to dissolved PLCL (70:30) in 1,4-dioxane. A 3D porous Coll/HA/PLCL scaffold was subsequently produced through freeze-drying/lyophilization and salt-leaching procedures. The resulting Coll/HA/PLCL scaffold displayed a high uniform porosity and highly interconnected pores. X-ray photoelectron spectrometer and Fourier transform infrared analyses revealed the presence of both collagen and HA particles on the surface of the Coll/HA/PLCL scaffold. Proliferation assay, microscopic observations, and gene analysis with quantitative RT-PCR showed that osteoblast cells were able to attach, proliferate, and maintain an osteoblastlike phenotype when cultured on the Coll/HA/PLCL scaffold. In summary, we produced a nontoxic scaffold that contains natural polymers (Coll and HA) and synthetic polymer (PLCL). Through its chemical composition and porous morphology, this scaffold may be useful for osteoblast growth, differentiation, and bone tissue formation.     

Response of human bone marrow stromal cells to a resorbable P2O5–SiO2–CaO–MgO–Na2O–K2O phosphate glass ceramic for tissue engineering applications

This work focuses on the synthesis and characterization of a novel bioresorbable glass ceramic phosphate-based material (GC-ICEL). More specifically, its solubility in different aqueous media (water, Tris–HCl and acellular simulated body fluid) and the response of human stromal cells cultured on it were investigated. X-ray diffraction analysis showed the presence of two crystalline phases identified as Na₂Mg(PO₄)₃ and Ca₂P₂O₇ and dissolution tests highlighted a preferential dissolution of the Na₂Mg(PO₄)₃ phase and of the residual amorphous phase in all the chosen media. Soaking tests in simulated body fluid showed precipitation of a hydroxyapatite layer, demonstrating the bioactivity of GC-ICEL, which is partially due to the reported bioactivity of Ca₂P₂O₇. The effect of GC-ICEL on adhesion, proliferation and osteoblastic gene expression of human bone marrow-derived stromal cells was also studied. Combining molecular and biochemical analyses, it was found that bone marrow cell differentiation was stimulated over proliferation on GC-ICEL. Moreover, the expression of bone-related genes in cells cultured on GC-ICEL confirmed the bioactivity of this phosphate-based glass ceramic, which might have a stimulatory effect on osteogenesis.     

The enhancement of bone regeneration by a combination of osteoconductivity and osteostimulation using β-CaSiO3/β-Ca3(PO4)2 composite bioceramics

β-Tricalcium phosphate (β-TCP) is osteoconductive, while β-calcium silicate (β-CS) is bioactive with osteostimulative properties. Porous β-CaSiO₃/β-Ca₃(PO₄)₂ composite bioceramic scaffolds with various β-TCP:β-CS ratios were designed to combine both osteoconductivity and osteostimulation in order to enhance bone regeneration. The composite scaffolds were implanted in critical sized femur defects (6 × 12 mm) for 4, 12 and 26 weeks with pure β-TCP and β-CS scaffolds as the controls. The in vivo biodegradation and bone regeneration of the specimens were investigated using sequential histological evaluations, immunohistochemical examination and micro-computed tomography technology. The results showed that the scaffolds with 50 and 80 wt.% β-CS dramatically enhanced the amount of newly formed bone and reduced the degradation rate. In contrast, porous β-CS displayed poor new bone formation due to its rapid degradation, while porous β-TCP showed moderate bone regeneration starting on the surface of the implants, due to a lack of osteostimulation. More importantly, the scaffolds with 50 and 80 wt.% β-CS not only had excellent osteoconductivity, but also stimulated rapid bone formation, and they could degrade progressively at a rate matching the regeneration of new bone. In summary, our findings indicated that the degradation rate and bioactivity of β-CS/β-TCP composite bioceramic scaffolds could be adjusted by controlling the ratio of β-CS to β-TCP, suggesting the potential application of β-CS/β-TCP composite bioceramic scaffolds with 50 and 80 wt.% β-CS component in hard tissue regeneration and bone tissue engineering.     

胰岛素生长因子1(IGF-1)通过S1P/S1PR1信号激活PI3K通路促进小鼠肺泡上皮细胞迁移

目的 探讨胰岛素样生长因子1(IGF-1)对肺泡上皮细胞(AEC)迁移的影响及其相关机制.方法 在磷脂酰肌醇3激酶(PI3K)抑制剂渥曼青霉素(Wortmannin)存在与否的情况下,采用IGF-1、鞘氨醇1磷酸(S1P)刺激AEC后,划痕愈合实验检测MLE-12小鼠AEC的迁移,Western blot法检测磷酸化的...