Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Abstract

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

可降解水凝胶作为关节软骨修复材料的研究进展

可降解水凝胶因其良好的生物相容性和生物降解性被广泛用于关节软骨的修复和再生。本文以可降解水凝胶在软骨组织工程中的三类应用策略为主线,概述了用于原位成型可注射水凝胶的蛋白多糖类材料及纳米复合类材料;系统总结了传统工艺制造组织工程支架的优缺点及多种工艺结合的制备方法;重点归纳了近年来3D打印组织工程支架从纯软骨到骨/软骨一体化、从单层到多层的研究进展;最后分析了可降解水凝胶作为关节软骨支架材料在微观定向结构和生物活性功能化方面的局限性,并作出展望：未来开展多材料、多尺度、多诱导的高仿生梯度支架是关节软骨组织工程的一个重要研究方向。     

Deposition behavior and properties of silk fibroin scaffolds soaked in simulated body fluid

Porous 3D silk fibroin (SF) scaffolds were prepared directly from the SF solution with the addition of methanol and glutaraldehyde by a freeze-drying method. The scaffolds were then soaked in the simulated body fluid (SBF) for the deposition of hydroxyapatite (HA) crystals. The XRD and FTIR results showed that the SF were in β-sheet structure, resulting in the high thermal stability and mechanical properties of scaffolds. The XRD and AAS data revealed that the SF scaffolds could induce the continuous growth and enrichment of HA crystals onto the scaffolds with the extension of soaking time. The mechanical properties of scaffolds increased first with the HA-deposition within 3 d of soaking, then it declined. During the full soaking period, no significant change was observed on the porosity and water-binding ability, which were kept at about 84% and 800%, respectively. The cell cultivation results showed that the scaffolds have the satisfied cell biocompatibility, which was promoted after the HA-deposition. This work suggests that the porous 3D SF scaffolds may be a potential candidate in the bone engineering.     

Immobilization of gelatin on bacterial cellulose nanofibers surface via crosslinking technique

Bacterial cellulose is considered to be a potential material for tissue engineering. However, the absence of enough activity restricts its practical application as tissue engineering scaffold. This paper describes the synthesis of a novel bacterial cellulose/gelatin composite via crosslinking by procyanidin (PA). The morphology of the bacterial cellulose/gelatin composite was observed by field emission scanning electron microscopy (FE-SEM) and transmission electronic microscope (TEM). The composites were further characterized by fourier transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD). It was found that the 0.25 wt.% Gel solution was the appropriate concentration for the BC/Gel composite. Furthermore, the proliferation, infiltration and adhesion of NIH3T3 cells on the BC/Gel-025 composite were evaluated. The results showed that the composite had better bioactivity than pure bacterial cellulose, and the composite supported cell growth.     

二氧化锆掺入对三维打印的硅酸二钙支架物理化学和生物学性能的影响

三维打印结合高分子前躯体制备生物陶瓷材料由于制备工艺简单, 在骨组织工程修复领域引起了极大的关注。本文成功利用三维打印技术与高分子硅胶前躯体结合, 通过填充活性CaCO₃和惰性ZrO₂制备出ZrO₂掺杂的β-Ca₂SiO₄支架。制备得到的支架具有均一、连通的大孔结构(孔隙率>67%), 随着掺杂ZrO₂含量的增加, 支架的抗压强度明显提高, 并且促进成骨细胞增殖、分化。重要的是在动物体内实验发现, 相较于纯的β-Ca₂SiO₄支架, ZrO₂的掺入明显提高了支架在骨缺损处促进新骨形成的能力。因而, 通过三维打印结合高分子前躯体技术制备掺杂ZrO₂的β-Ca₂SiO₄支架有望应用于骨组织工程。     

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.     

β-CaSiO_3/丝素蛋白复合支架材料结构与性能的研究

利用冷冻干燥法制备了β-CaSiO_3/丝素蛋白复合支架材料,经XRD和FTIR分析表明复合支架中丝素的结构主要以β-折叠为主;SEM分析显示材料孔隙分布均匀,孔连通性较好,孔径尺寸约为100～300μm.对支架的孔隙率和机械强度等性能进行了表征,研究表明复合支架的孔隙率为83%～87%,机械强度有较大提高.应用模拟体液浸泡实验研究了复合支架的体外生物活性,并用XRD、FESEM和EDS对试样表面进行了表征;结果显示,样品经模拟体液浸泡3天后,表面都能沉积出类骨羟基磷灰石(HA)层,β-CaSiO_3的加入能加快复合支架表面沉积类骨HA的速度.研究结果显示β-CaSiO_3/丝素蛋白复合支架材料有望作为强度较好的生物活性硬组织修复材料.     

In vitro biodegradation and biocompatibility of gelatin/montmorillonite-chitosan intercalated nanocomposite

The intercalated nanocomposite of gelatin/montmorillonite-chitosan (Gel/MMT-CS) was prepared via the solution intercalation process. In vitro degradation tests showed that the nanocomposite had a lower degradation rate than Gel-CS composite. And the introduced intercalation structure endowed Gel/MMT-CS nanocomposite with a controllable degradation rate when changing the MMT content. Cells attachment, spread and proliferation on the Gel/MMT-CS membranes were investigated by scanning electron microscopy (SEM) and mitochondrial activity assay. The results provided evidences of good adhesion, proliferation and morphology of rat stromal stem cells on Gel/MMT-CS membranes compared to the tissue culture plates (TCPs), making the Gel/MMT-CS nanocomposite a promising candidate towards tissue engineering.     

Chitosan/gelatin scaffolds support bone regeneration

Chitosan/Gelatin (CS:Gel) scaffolds were fabricated by chemical crosslinking with glutaraldehyde or genipin by freeze drying. Both crosslinked CS:Gel scaffold types with a mass ratio of 40:60% form a gel-like structure with interconnected pores. Dynamic rheological measurements provided similar values for the storage modulus and the loss modulus of the CS:Gel scaffolds when crosslinked with the same concentration of glutaraldehyde vs. genipin. Compared to genipin, the glutaraldehyde-crosslinked scaffolds supported strong adhesion and infiltration of pre-osteoblasts within the pores as well as survival and proliferation of both MC3T3-E1 pre-osteoblastic cells after 7 days in culture, and human bone marrow mesenchymal stem cells (BM-MSCs) after 14 days in culture. The levels of collagen secreted into the extracellular matrix by the pre-osteoblasts cultured for 4 and 7 days on the CS:Gel scaffolds, significantly increased when compared to the tissue culture polystyrene (TCPS) control surface. Human BM-MSCs attached and infiltrated within the pores of the CS:Gel scaffolds allowing for a significant increase of the osteogenic gene expression of RUNX2, ALP, and OSC. Histological data following implantation of a CS:Gel scaffold into a mouse femur demonstrated that the scaffolds support the formation of extracellular matrix, while fibroblasts surrounding the porous scaffold produce collagen with minimal inflammatory reaction. These results show the potential of CS:Gel scaffolds to support new tissue formation and thus provide a promising strategy for bone tissue engineering.     

3D printed porous β-Ca2SiO4 scaffolds derived from preceramic resin and their physicochemical and biological properties

Silicate bioceramic scaffolds are of great interest in bone tissue engineering, but the fabrication of silicate bioceramic scaffolds with complex geometries is still challenging. In this study, three-dimensional (3D) porous β-Ca₂SiO₄ scaffolds have been successfully fabricated from preceramic resin loaded with CaCO₃ active filler by 3D printing. The fabricated β-Ca₂SiO₄ scaffolds had uniform interconnected macropores (ca. 400 μm), high porosity (>78%), enhanced mechanical strength (ca. 5.2 MPa), and excellent apatite mineralization ability. Importantly, the results showed that the increase of sintering temperature significantly enhanced the compressive strength and the scaffolds sintered at higher sintering temperature stimulated the adhesion, proliferation, alkaline phosphatase activity, and osteogenic-related gene expression of rat bone mesenchymal stem cells. Therefore, the 3D printed β-Ca₂SiO₄ scaffolds derived from preceramic resin and CaCO₃ active fillers would be promising candidates for bone tissue engineering.     

Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility

To construct a novel scaffold for nucleus pulposus (NP) tissue engineering, The porous type II collagen (CII)/hyaluronate (HyA)–chondroitin-6-sulfate (6-CS) scaffold was prepared using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking system. The physico-chemical properties and biocompatibility of CII/HyA–CS scaffolds were evaluated. The results suggested CII/HyA–CS scaffolds have a highly porous structure (porosity: 94.8 ± 1.5%), high water-binding capacity (79.2 ± 2.8%) and significantly improved mechanical stability by EDC/NHS crosslinking (denaturation temperature: 74.6 ± 1.8 and 58.1 ± 2.6°C, respectively, for the crosslinked scaffolds and the non-crosslinked; collagenase degradation rate: 39.5 ± 3.4 and 63.5 ± 2.0%, respectively, for the crosslinked scaffolds and the non-crosslinked). The CII/HyA–CS scaffolds also showed satisfactory cytocompatibility and histocompatibility as well as low immunogenicity. These results indicate CII/HyA–CS scaffolds may be an alternative material for NP tissue engineering due to the similarity of its composition and physico-chemical properties to those of the extracellular matrices (ECM) of native NP.     

静电纺丝天然-人工合成聚合物复合纳米纤维及其对细胞黏附、增殖和迁移的影响

生物材料组成成分对细胞生物功能有不同的影响。利用静电纺丝技术制备了基于聚己内酯(PCL,polycaprolactone)的不同天然蛋白、多糖(丝素蛋白(SF,silk fibroin)、透明质酸(HA,hyaluronicacid))的混合组分纳米纤维,采用了扫描电镜和接触角对纳米纤维进行基础表征。同时,进一步考察了纳米纤维作为组织工程支架的可行性。研究结果表明SF组分能增加材料的可纺性,有利于细胞的前期黏附,并能够促进细胞增殖。HA组分可以改善材料的亲水性,增加细胞伪足并促进细胞迁移。重要的是,PCL/SF/HA纳米纤维能同时结合SF和HA的优点,有望在组织工程领域得到应用。     

Modification of human cancellous bone using Thai silk fibroin and gelatin for enhanced osteoconductive potential

The modification of human cancellous bone (hBONE) with silk fibroin/gelatin (SF/G) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccini-mide (NHS) crosslinking was established. The SF/G solutions at a weight ratio of 50/50 and the solution concentrations of 1, 2, and 4 wt % were studied. SF/G sub-matrix was formed on the surface and inside pore structure of hBONE. All hBONE scaffolds modified with SF/G showed smaller pore sizes, less porosity, and slightly lower compressive modulus than unmodified hBONE. SF/G sub-matrix was gradually biodegraded in collagenase solution along 4 days. The hBONE scaffolds modified with SF/G, particularly at 2 and 4 wt % solution concentrations, promoted attachment, proliferation, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSC), comparing to the original hBONE. The highest cell number, ALP activity and calcium production were observed for MSC cultured on the hBONE scaffolds modified with 4 wt % SF/G. The mineralization was also remarkably induced in the cases of modified hBONE scaffolds as observed from the deposited calcium phosphate by EDS. The modification of hBONE with SF/G was, therefore, the promising method to enhance the osteoconductive potential of human bone graft for bone tissue engineering.     

Fabrication of nanofibrous macroporous scaffolds of poly(lactic acid) incorporating bioactive glass nanoparticles by camphene-assisted phase separation

Here we produced macroporous and nanofibrous scaffolds with bioactive nanocomposite composition, poly(lactic acid) (PLA) incorporating bioactive glass nanoparticles (BGnp) up to 30 wt%, targeting bone regeneration. In particular, the nanofibrous structure in the scaffolds was generated by using a bicyclic monoterpene, camphene (C₁₀H₁₆), through a phase-separation process with PLA-BGnp phase in chloroform/1,4-dioxane co-solvent. Furthermore, macropores were produced by the impregnation of salt particles and their subsequent leaching out, followed by freezing and lyophilization processes. The produced PLA-BGnp scaffolds presented highly porous and nanofibrous structure with porosities of 90–95% and pore sizes of over hundreds of micrometers. BGnp with sizes of ∼90 nm were also evenly impregnated within the PLA matrix, featuring a nanocomposite structure. The nanofibrous scaffolds exhibited enhanced hydrophilicity and more rapid hydrolytic degradation as the incorporated BGnp content increased. The bone-bioactivity of the scaffolds was substantially improved with the incorporation of BGnp, exhibiting rapid formation of apatite throughout the scaffolds in a simulated body fluid. The developed macroporous and nanofibrous scaffolds with PLA-BGnp bioactive composition are considered as a novel 3D matrix potentially useful for bone tissue engineering.     

Fabrication and in vitro evaluations with osteoblast-like MG-63 cells of porous hyaluronic acid-gelatin blend scaffold for bone tissue engineering applications

In this study, hyaluronic acid–gelatin (HyA–Gel) scaffolds were prepared with HyA:Gel ratios of 15:85, 50:50, and 85:15 with the goal of obtaining a porous biocompatible scaffold for bone tissue engineering applications. Scanning electron microscopy and Fourier-transform infrared spectroscopy were done to characterize the morphological orientations of the scaffolds. The biocomposite structure was highly porous and the pores in the scaffolds were interconnected. The compressive strength of the scaffold was 7.39 ± 0.2 MPa for the HyA–Gel when fabricated at a ratio of 15:85. To assess the biocompatibility and cell behavior on the HyA–Gel biocomposite, the proliferation of MG-63 osteoblast cell on the scaffolds was examined using the MTT assay, optical microscopy, and confocal microscopy. Collagen type I and osteopontin expression of cells cultured on the scaffolds were examined using immunoblotting. The scaffolds fabricated with a 15:85—HyA:Gel ratio showed excellent biocompatibility, good mechanical properties, and high porosity, which suggest that the highly porous scaffold holds great promise for use in bone tissue engineering applications.     

A review on powder-based additive manufacturing for tissue engineering: selective laser sintering and inkjet 3D printing

Abstract

Since most starting materials for tissue engineering are in powder form, using powder-based additive manufacturing methods is attractive and practical. The principal point of employing additive manufacturing (AM) systems is to fabricate parts with arbitrary geometrical complexity with relatively minimal tooling cost and time. Selective laser sintering (SLS) and inkjet 3D printing (3DP) are two powerful and versatile AM techniques which are applicable to powder-based material systems. Hence, the latest state of knowledge available on the use of AM powder-based techniques in tissue engineering and their effect on mechanical and biological properties of fabricated tissues and scaffolds must be updated. Determining the effective setup of parameters, developing improved biocompatible/bioactive materials, and improving the mechanical/biological properties of laser sintered and 3D printed tissues are the three main concerns which have been investigated in this article.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Silk fibroin-polyurethane scaffolds for tissue engineering

Silk fibroin (SF) is a highly promising protein for its surface and structural properties, associated with a good bio- and hemo-compatibility. However, its mechanical properties and architecture cannot be easily tailored to meet the requirements of specific applications. In this work, SF was used to modify the surface properties of polyurethanes (PUs), thus obtaining 2D and 3D scaffolds for tissue regeneration. PUs were chosen for their well known advantageous properties and versatility; they can be obtained either as 2D (films) or 3D (foams) substrates. Films of a medical-grade poly-carbonate-urethane were prepared by solvent casting; PU foams were purposely designed and prepared with a morphology (porosity and cell size) adequate for cell growth. PU substrates were coated with fibroin by a dipping technique. To stabilize the coating layer, a conformational change of the protein from the alpha-form (water soluble) to the beta-form (not water soluble) was induced. Novel methodology in UV spectroscopy were developed for quantitatively analyzing the SF-concentration in dilute solutions. Pure fibroin was used as standard, as an alternative to the commonly used albumin, allowing real concentration values to be obtained. SF-coatings showed good stability in physiological-like conditions. A treatment with methanol further stabilized the coating. Preliminary results with human fibroblasts indicated that SF coating promote cell adhesion and growth, suggesting that SF-modified PUs appear to be suitable scaffolds for tissue engineering applications.     

A review on powder-based additive manufacturing for tissue engineering: selective laser sintering and inkjet 3D printing

Since most starting materials for tissue engineering are in powder form, using powder-based additive manufacturing methods is attractive and practical. The principal point of employing additive manufacturing (AM) systems is to fabricate parts with arbitrary geometrical complexity with relatively minimal tooling cost and time. Selective laser sintering (SLS) and inkjet 3D printing (3DP) are two powerful and versatile AM techniques which are applicable to powder-based material systems. Hence, the latest state of knowledge available on the use of AM powder-based techniques in tissue engineering and their effect on mechanical and biological properties of fabricated tissues and scaffolds must be updated. Determining the effective setup of parameters, developing improved biocompatible/bioactive materials, and improving the mechanical/biological properties of laser sintered and 3D printed tissues are the three main concerns which have been investigated in this article.     

Reduced graphene oxide: osteogenic potential for bone tissue engineering

Collagen (Col) type I, as the major component of the bone extracellular matrix has been broadly studied for bone tissue engineering. However,inferior mechanical properties limit its usage for load bearing applications. In this research, freeze dried Col scaffolds are coated with graphene oxide (GO) through a covalent bond of the amine Col with the graphene carboxyl groups. The prepared scaffolds were then reduced using a chemical agent. Scanning electron microscopy exhibited a porous structure for the synthesized scaffolds with an approximate pore size of 100–220 ± 12 µm, which is in the suitable range for bone tissue engineering application. Reducing the GO coating improved the compressive modulus of the Col from 250 to 970 kPa. Apatite formation was also indicated by immersing the scaffolds in simulated body fluid after five days. The cytocompatibility of the scaffolds, using human bone marrow‐derived mesenchymal stem cells, was confirmed with MTT analysis. Alkaline phosphatase assay revealed that reducing the Col–GO scaffolds can effectively activate the differentiation of hBM‐MSCs into osteoblasts after 14 days, even without the addition of an osteogenic differentiation medium. The results of this study highlight that GO and its reduced form have considerable potential as bone substitutes for orthopaedic and dental applications.Inspec keywords: molecular biophysics, tissue engineering, biochemistry, cellular biophysics, graphene, biomedical materials, bone, proteins, scanning electron microscopy, porous materials, compressive strength, biomechanicsOther keywords: human bone marrow‐derived mesenchymal stem cells, reduced graphene oxide, bone extracellular matrix, inferior mechanical properties, load bearing applications, freeze‐dried Col scaffolds, amine Col groups, graphene carboxyl groups, bone tissue engineering, collagen type I, GO‐Col scaffolds, covalent bond, scanning electron microscopy, compressive modulus, apatite formation, cytocompatibility, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide analysis, alkaline phosphatase assay, osteogenic differentiation medium, dental applications, orthopaedic applications, porous structure, time 14.0 day, CO