Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110 μm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40 kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.     

溶剂浇铸/颗粒沥滤技术制备组织工程支架材料

生物可降解多孔三维细胞支架是组织工程化组织构建的基础。溶剂浇铸粒子沥滤技术是最简便、也是研究最广泛的一种多孔三维细胞支架制备技术,随着各种改进方法的出现,溶剂浇铸粒子沥滤已成为组织工程用多孔三维细胞支架的理想制备技术 。     

Enzymatic cross-linking versus radical polymerization in the preparation of gelatin PolyHIPEs and their performance as scaffolds in the culture of hepatocytes

Highly open porous biodegradable scaffolds, based on gelatin A3, were fabricated with the aim of using them for tissue-engineering applications. The fabrication process is based on an emulsion-templating technique. In the preparation of gelatin scaffolds two different cross-linking procedures were adopted: (I) radical polymerization of the methacrylate functionalities, previously introduced onto the gelatin chains and (II) formation of isopeptide bridges among the gelatin chains promoted by the enzyme microbial transglutaminase. The method of cross-linking exerts a pronounced effect on the morphology of the porous biomaterials: radical polymerization of methacrylated gelatin allowed the production of scaffolds with a better defined porous structure, while the enzymatically cross-linked scaffolds were characterized by a thinner skeletal framework. A suitable sample of each kind of the differently cross-linked porous biomaterials was tested for the culture of hepatocytes. The scaffold obtained by radical polymerization possessed a morphology characterized by relatively large voids and interconnects, and as a consequence, it was more suitable for hepatocytes colonization. On the other hand, the enzymatically cross-linked scaffold resulted in less cytotoxicity and the cultured hepatocytes expressed a better differentiated phenotype, as evidenced by a greater expression and more correct localization of key adhesion proteins.     

Bio-composite scaffolds containing chitosan/nano-hydroxyapatite/nano-copper-zinc for bone tissue engineering

The current study involves fabrication and characterization of bio-composite scaffolds containing chitosan (CS), nano-hydroxyapatite (nHAp) and Cu-Zn alloy nanoparticles (nCu-Zn) by freeze drying technique. The fabricated composite scaffolds (CS/nHAp and CS/nHAp/nCu-Zn) were characterized by SEM, EDX, XRD and FT-IR studies. The addition of nCu-Zn in the CS/nHAp scaffolds significantly increased swelling, decreased degradation, increased protein adsorption, and increased antibacterial activity. The CS/nHAp/nCu-Zn scaffolds had no toxicity towards rat osteoprogenitor cells. So the developed CS/nHAp/nCu-Zn scaffolds have advantageous and potential applications over the CS-nHAp scaffolds for bone tissue engineering.     

Supercritical fluid-assisted controllable fabrication of open and highly interconnected porous scaffolds for bone tissue engineering

Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional(3 D)scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone(PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 2~3 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2(BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells(BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase(ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.     

Living bacterial sacrificial porogens to engineer decellularized porous scaffolds

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types.     

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity¹. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes². However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia^3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries^5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)⁷ for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.     

Silk scaffolds connected with different naturally occurring biomaterials for prostate cancer cell cultivation in 3D

In the present work, different biopolymer blend scaffolds based on the silk protein fibroin from Bombyx mori (BM) were prepared via freeze‐drying method. The chemical, structural, and mechanical properties of the three dimensional (3D) porous silk fibroin (SF) composite scaffolds of gelatin, collagen, and chitosan as well as SF from Antheraea pernyi (AP) and the recombinant spider silk protein spidroin (SSP1) have been systematically investigated, followed by cell culture experiments with epithelial prostate cancer cells (LNCaP) up to 14 days. Compared to the pure SF scaffold of BM, the blend scaffolds differ in porous morphology, elasticity, swelling behavior, and biochemical composition. The new composite scaffold with SSP1 showed an increased swelling degree and soft tissue like elastic properties. Whereas, in vitro cultivation of LNCaP cells demonstrated an increased growth behavior and spheroid formation within chitosan blended scaffolds based on its remarkable porosity, which supports nutrient supply matrix. Results of this study suggest that silk fibroin matrices are sufficient and certain SF composite scaffolds even improve 3D cell cultivation for prostate cancer research compared to matrices based on pure biomaterials or synthetic polymers.     

快速成形技术制造组织工程支架研究进展

支架作为组织工程的关键要素之一, 影响着所接种细胞的分布和增值以及新组织的形成。传统的方法虽然可以制造出各种孔隙率的支架, 但缺乏对支架多孔结构的控制。近年来, 快速成形技术发展迅速, 并成功应用于组织工程支架的制造, 实现了组织工程支架内部多孔结构与复杂外形的精确控制, 从而使得构建理想的组织工程化结构体成为可能。以下回顾了应用快速成形技术制造组织工程支架的优势与潜力, 展望了未来组织工程支架的设计制造发展方向。     

Processing/structure/property relationship of multi-scaled PCL and PCL-HA composite scaffolds prepared via gas foaming and NaCl reverse templating

In this study, we investigated the processing/structure/property relationship of multi-scaled porous biodegradable scaffolds prepared by combining the gas foaming and NaCl reverse templating techniques. Poly(ε-caprolactone) (PCL), hydroxyapatite (HA) nano-particles and NaCl micro-particles were melt-mixed by selecting different compositions and subsequently gas foamed by a pressure-quench method. The NaCl micro-particles were finally removed from the foamed systems in order to allow for the achievement of the multi-scaled scaffold pore structure. The control of the micro-structural properties of the scaffolds was obtained by the optimal combination of the NaCl templating concentration and the composition of the CO2-N2 mixture as the blowing agent. In particular, these parameters were accurately selected to allow for the fabrication of PCL and PCL-HA composite scaffolds with multi-scaled open pore structures. Finally, the biocompatibility of the scaffolds has been assessed by cultivating pre-osteoblast MG63 cells in vitro, thus demonstrating their potential applications for bone regeneration.     

Melatonin delivery from PCL scaffold enhances glycosaminoglycans deposition in human chondrocytes – Bioactive scaffold model for cartilage regeneration

The therapeutic potential of an engineered cartilage construct can be enhanced by sustained delivery of chondrogenic drug like melatonin from 3D porous scaffolds embedded with melatonin loaded bovine serum albumin nanoparticles (MNP). In this study, MNP was synthesized and loaded into polycaprolactone (PCL) scaffolds. 12 % (w/v) and 10 % (w/v) PCL scaffolds were fabricated with different concentrations of MNP. X- ray diffraction and Raman analysis of MNP and scaffolds revealed amorphization of melatonin which is highly desired in drug delivery applications. Additionally, Fourier Transform Infrared spectroscopic analysis confirmed the drug to be chemically inert to fabrication process. Field emission scanning electron microscopic analysis suggested highly interlinked porous scaffold (diameter 50 μm – 300 μm) and MNP diameters in the range of 110−200 nm. Importantly, UV spectrophotometric analysis showed that all groups of scaffolds showed sustained release for 21 days, wherein MNP concentration had an influence on release behaviour of melatonin from scaffolds. Drug release kinetics studied using mathematical models revealed, diffusion and dissolution mechanism of release. Furthermore, in vitro evaluation of MNP loaded scaffolds with Human chondrocytes for 21 days increased glycosaminoglycans deposition significantly. In brief, sustained release of melatonin from polycaprolactone scaffolds increased the therapeutic potential of the engineered construct.     

Fabrication of three-dimensional collagen scaffold using an inverse mould-leaching process

Natural biopolymers, such as collagen or chitosan, are considered ideal for biomedical scaffolds. However, low processability of the materials has hindered the fabrication of designed pore structures controlled by various solid freeform-fabrication methods. A new technique to fabricate a biomedical three-dimensional collagen scaffold, supplemented with a sacrificial poly(ethylene oxide) mould is proposed. The fabricated collagen scaffold shows a highly porous surface and a three-dimensional structure with high porosity as well as mechanically stable structure. To show its feasibility for biomedical applications, fibroblasts/keratinocytes were co-cultured on the scaffold, and the cell proliferation and cell migration of the scaffold was more favorable than that obtained with a spongy-type collagen scaffold.     

Effect of Layer Thickness and Printing Orientation on Mechanical Properties and Dimensional Accuracy of 3D Printed Porous Samples for Bone Tissue Engineering

Powder-based inkjet 3D printing method is one of the most attractive solid free form techniques. It involves a sequential layering process through which 3D porous scaffolds can be directly produced from computer-generated models. 3D printed products'' quality are controlled by the optimal build parameters. In this study, Calcium Sulfate based powders were used for porous scaffolds fabrication. The printed scaffolds of 0.8 mm pore size, with different layer thickness and printing orientation, were subjected to the depowdering step. The effects of four layer thicknesses and printing orientations, (parallel to X, Y and Z), on the physical and mechanical properties of printed scaffolds were investigated. It was observed that the compressive strength, toughness and Young''s modulus of samples with 0.1125 and 0.125 mm layer thickness were more than others. Furthermore, the results of SEM and μCT analyses showed that samples with 0.1125 mm layer thickness printed in X direction have more dimensional accuracy and significantly close to CAD software based designs with predefined pore size, porosity and pore interconnectivity.     

Polyphosphazene/nano-hydroxyapatite composite microsphere scaffolds for bone tissue engineering

The nontoxic, neutral degradation products of amino acid ester polyphosphazenes make them ideal candidates for in vivo orthopedic applications. The quest for new osteocompatible materials for load bearing tissue engineering applications has led us to investigate mechanically competent amino acid ester substituted polyphosphazenes. In this study, we have synthesized three biodegradable polyphosphazenes substituted with side groups, namely, leucine, valine, and phenylalanine ethyl esters. Of these polymers, the phenylalanine ethyl ester substituted polyphosphazene showed the highest glass transition temperature (41.6 degrees C) and, hence, was chosen as a candidate material for forming composite microspheres with 100 nm sized hydroxyapatite (nHAp). The fabricated composite microspheres were sintered into a three-dimensional (3-D) porous scaffold by adopting a dynamic solvent sintering approach. The composite microsphere scaffolds showed compressive moduli of 46-81 MPa with mean pore diameters in the range of 86-145 microm. The 3-D polyphosphazene-nHAp composite microsphere scaffolds showed good osteoblast cell adhesion, proliferation, and alkaline phosphatase expression and are potential suitors for bone tissue engineering applications.     

Evaluation of Biological Properties of Electron Beam Melted Ti6Al4V Implant with Biomimetic Coating In Vitro and In Vivo

Background

High strength porous titanium implants are widely used for the reconstruction of craniofacial defects because of their similar mechanical properties to those of bone. The recent introduction of electron beam melting (EBM) technique allows a direct digitally enabled fabrication of patient specific porous titanium implants, whereas both their in vitro and in vivo biological performance need further investigation.

Methods

In the present study, we fabricated porous Ti6Al4V implants with controlled porous structure by EBM process, analyzed their mechanical properties, and conducted the surface modification with biomimetic approach. The bioactivities of EBM porous titanium in vitro and in vivo were evaluated between implants with and without biomimetic apatite coating.

Results

The physical property of the porous implants, containing the compressive strength being 163 - 286 MPa and the Young’s modulus being 14.5–38.5 GPa, is similar to cortical bone. The in vitro culture of osteoblasts on the porous Ti6Al4V implants has shown a favorable circumstance for cell attachment and proliferation as well as cell morphology and spreading, which were comparable with the implants coating with bone-like apatite. In vivo, histological analysis has obtained a rapid ingrowth of bone tissue from calvarial margins toward the center of bone defect in 12 weeks. We observed similar increasing rate of bone ingrowth and percentage of bone formation within coated and uncoated implants, all of which achieved a successful bridging of the defect in 12 weeks after the implantation.

Conclusions

This study demonstrated that the EBM porous Ti6Al4V implant not only reduced the stress-shielding but also exerted appropriate osteoconductive properties, as well as the apatite coated group. The results opened up the possibility of using purely porous titanium alloy scaffolds to reconstruct specific bone defects in the maxillofacial and orthopedic fields.     

天然聚合物多孔支架的冻干工艺研究

目的 :通过改变冷冻干燥的工艺条件 ,缩短制备聚合物多孔支架的时间。方法 :把冷冻好的聚合物溶液在溶剂的冰点以上直接真空升华干燥 ,通过对支架的孔结构和外观的考察 ,评价其作为制备多孔支架工艺的可行性。结果 :制备了壳聚糖、胶原、明胶 3种不同材料的支架 ,都具有均匀的通孔结构 ,可应用于组织工程研究。结论 :在比冰点高的温度下对冷冻的样品进行真空干燥 ,可以制得多孔支架 ,并能使制备的时间大大缩短 ,但该工艺方案有时会产生开裂和塌陷 ,影响支架的质量 ,因此还有待进一步完善。     

Biological properties of solid free form designed ceramic scaffolds with BMP-2: in vitro and in vivo evaluation

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.     

Advances in polymeric systems for tissue engineering and biomedical applications

The characteristics of tissue engineered scaffolds are major concerns in the quest to fabricate ideal scaffolds for tissue engineering applications. The polymer scaffolds employed for tissue engineering applications should possess multifunctional properties such as biocompatibility, biodegradability and favorable mechanical properties as it comes in direct contact with the body fluids in vivo. Additionally, the polymer system should also possess biomimetic architecture and should support stem cell adhesion, proliferation and differentiation. As the progress in polymer technology continues, polymeric biomaterials have taken characteristics more closely related to that desired for tissue engineering and clinical needs. Stimuli responsive polymers also termed as smart biomaterials respond to stimuli such as pH, temperature, enzyme, antigen, glucose and electrical stimuli that are inherently present in living systems. This review highlights the exciting advancements in these polymeric systems that relate to biological and tissue engineering applications. Additionally, several aspects of technology namely scaffold fabrication methods and surface modifications to confer biological functionality to the polymers have also been discussed. The ultimate objective is to emphasize on these underutilized adaptive behaviors of the polymers so that novel applications and new generations of smart polymeric materials can be realized for biomedical and tissue engineering applications.     

The development and identification of constructing tissue engineered bone by seeding osteoblasts from differentiated rat marrow stromal stem cells onto three-dimensional porous nano-hydroxylapatite bone matrix in vitro

The purposes of this study were to develop a new cultural method for the rat bone marrow stromal cells (MSCs) to differentiate into osteoblasts well in vitro, and to investigate the feasibility of using MSCs as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds for constructing tissue-engineered bone. MSCs of rats were isolated, cultured, induced to differentiate into osteoblasts, and then observed with inverted microscopy. Histochemical staining and radio-immunological analysis were applied for identifying MSCs. Whereafter MSCs were seeded onto three-dimensional porous nano-hydroxylapatite scaffolds, and scanning electron microscopy was applied to evaluate their growth on scaffolds. Results showed that MSCs were typical fibroblast-like and possessed a better proliferating capability; the activity of alkaline phosphatase (ALP) and the secretion of osteocalcin of MSCs were produced gradually and increased continuously; the cells seeded on three-dimensional porous nano-hydroxylapatite scaffolds adhered, proliferated and differentiated well. These results demonstrated that the new improved culture method had the advantages of short isolating time, less risk of contamination and higher efficiency and accordingly was conducive to MSCs proliferating and differentiating into osteoblasts, and that it was advantageous to constructing tissue-engineered bone using MSCs as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds.     

Porous 3-D scaffolds from regenerated silk fibroin

Three fabrication techniques, freeze-drying, salt leaching and gas foaming, were used to form porous three-dimensional silk biomaterial matrixes. Matrixes were characterized for morphological and functional properties related to processing method and conditions. The porosity of the salt leached scaffolds varied between 84 and 98% with a compressive strength up to 175 +/- 3 KPa, and the gas foamed scaffolds had porosities of 87-97% and compressive strength up to 280 +/- 4 KPa. The freeze-dried scaffolds were prepared at different freezing temperatures (-80 and -20 degrees C) and subsequently treated with different concentrations (15 and 25%) and hydrophilicity alcohols. The porosity of these scaffolds was up to 99%, and the maximum compressive strength was 30 +/- 2 KPa. Changes in silk fibroin structure during processing to form the 3D matrixes were determined by FT-IR and XrD. The salt leached and gas foaming techniques produced scaffolds with a useful combination of high compressive strength, interconnected pores, and pore sizes greater than 100 microns in diameter. The results suggest that silk-based 3D matrixes can be formed for utility in biomaterial applications.