Novel 3D collagen scaffolds fabricated by indirect printing technique for tissue engineering

This article reports the mechanical properties and in vitro evaluation of a collagen scaffold fabricated using an indirect 3D printing technique. Collagen scaffolds, featuring predefined internal channels and capillary networks, were manufactured using phase change printing. It was observed that the collagen scaffolds featured internal channels and a hierarchical structure that varied over length scales of 10-400 microm. In vitro evaluation using hMSCs demonstrated that the resultant collagen based scaffolds have the ability to support hMSC cell attachment and proliferation; cells can migrate and survive deep within the structure of the scaffold. The cell numbers increased 2.4 times over 28 days in culture for the lysine treated scaffolds. The cells were spread along the collagen fibers to form a 3D structure and extracellular matrix was detected on the surface of the scaffolds after 4 weeks in culture. The crosslinking treatment enhanced the biostability and dynamic properties of the collagen scaffolds significantly.     

Physicomechanical properties of sintered scaffolds formed from porous and protein-loaded poly(DL-lactic-co-glycolic acid) microspheres for potential use in bone tissue engineering

An injectable poly(DL-lactic-co-glycolic acid) (PLGA) system comprising both porous and protein-loaded microspheres capable of forming porous scaffolds at body temperature was developed for tissue regeneration purposes. Porous and non-porous (lysozyme loaded) PLGA microspheres were formulated to represent ‘low molecular weight’ 22–34 kDa, ‘intermediate molecular weight’ (IMW) 53 kDa and ‘high molecular weight’ 84–109 kDa PLGA microspheres. The respective average size of the microspheres was directly related to the polymer molecular weight. An initial burst release of lysozyme was observed from both microspheres and scaffolds on day 1. In the case of the lysozyme-loaded microspheres, this burst release was inversely related to the polymer molecular weight. Similarly, scaffolds loaded with 1 mg lysozyme/g of scaffold exhibited an inverse release relationship with polymer molecular weight. The burst release was highest amongst IMW scaffolds loaded with 2 and 3 mg/g. Sustained lysozyme release was observed after day 1 over 50 days (microspheres) and 30 days (scaffolds). The compressive strengths of the scaffolds were found to be inversely proportional to PLGA molecular weight at each lysozyme loading. Surface analysis indicated that some of the loaded lysozyme was distributed on the surfaces of the microspheres and thus responsible for the burst release observed. Overall the data demonstrates the potential of the scaffolds for use in tissue regeneration.     

Biomimetic scaffolds fabricated from apatite-coated polymer microspheres

The deposition of a bonelike mineral on the surface of polymer scaffolds results in the formation of hybrid biomaterials, possessing enhanced osteoconductivity while retaining appropriate biodegradability. However, current methods of fabricating such composite scaffolds use a prolonged incubation process, which permits scaffold deformation and premature loss of incorporated macromolecules. We hypothesized that the fabrication of biomineralized polymer scaffolds could be achieved using premineralized polymer microspheres generated through incubation in a modified simulated body fluid (mSBF). We explored the material characteristics of these substrates and characterized the in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) when cultured on these novel scaffolds. Unlike scaffolds prepared using the conventional approach, premineralized scaffolds maintained their initial conformation after fabrication, achieved improved mineral distribution throughout the substrate, and enabled significantly greater incorporation efficiency of a model protein. We did not detect differences in osteogenic differentiation as determined by alkaline phosphatase activity and osteopontin secretion. However, we did observe a significant increase in cell-secreted calcium by hMSCs seeded on scaffolds prepared from premineralized polymer. These results demonstrate that the use of premineralized polymeric materials to fabricate biodegradable polymer scaffolds is an improved method for composite scaffold formation and may have numerous advantages for use in bone tissue engineering.     

Drug-releasing scaffolds fabricated from drug-loaded microspheres.

Biodegradable scaffolds serve a central role in many strategies for engineering tissue replacements or in guiding tissue regeneration. Typically, these scaffolds function to create and maintain a space and to provide a support for cell adhesion. However, these scaffolds also can serve as vehicles for the delivery of bioactive factors (e.g., protein or DNA) in order to manipulate cellular processes within the scaffold microenvironment. This study presents a novel approach to fabricate tissue-engineering scaffolds capable of sustained drug delivery whereby drug-loaded microspheres are fabricated into structures with controlled porosity. A double-emulsion process was used to fabricate microspheres with encapsulated DNA that retained its integrity and was released from the microspheres within 24 h. These DNA-loaded microspheres subsequently were formed into a nonporous disk or an interconnected open-pore scaffold (>94% porosity) via a gas-foaming process. The disks and scaffolds exhibited sustained plasmid release for at least 21 days and had minimal burst during the initial phase of release. This approach of assembling drug-loaded microspheres into porous and nonporous structures may find great utility in the fabrication of synthetic matrices that direct tissue formation.     

Novel fabricated matrix via electrospinning for tissue engineering

Electric field-driven fiber formation (electrospinning) is developing into a practical means for preparing novel porous filament with unusual structures and affordable mechanical properties. Polycaprolactone (PCL) was dissolved in solvent mixtures of methylene chloride/N,N-dimethyl formamide with ratios of 100/0, 75/25, and 50/50 (v/v) for electrospinning. The filament was formed by coagulation of the spinning solution following the well-known principle of phase separation in polymer solutions valid in other wet shaping processes. A strand of electrospun porous filament consisted of fibers ranging from 0.5 to 12 microm in diameter. To evaluate the feasibility of three-dimensional fabric as scaffold matrices, the plain weave, which is the simplest of the weaves and the most common, was prepared with porous PCL filament. The growth characteristics of MCF-7 mammary carcinoma cells in the woven fabrics showed the important role of matrix microstructure in proliferation. This study has shown that woven fabrics, consisting of porous filaments via electrospinning, may be suitable candidates as tissue engineering scaffolds.     

PCL-TCP wet spun scaffolds carrying antibiotic-loaded microspheres for bone tissue engineering

Abstract

Scaffolds produced for tissue engineering applications are proven to be promising alternatives to be used in healing and regeneration of injured tissues and organs. In this study, porous and fibrous poly(ε-caprolactone) (PCL) scaffolds were prepared by wet spinning technique and modified by addition of tricalcium phosphate (TCP) and by immobilizing gelatin onto fibers. Meanwhile, gelatin microspheres carrying Ceftriaxone sodium (CS), a model antibiotic, were added onto the scaffolds and antimicrobial activity of CS was investigated against Escherichia coli (E. coli), a model gram-negative bacterium. TCP and gelatin were added to enhance mechanical properties while directing the scaffold towards osteogenic infrastructure and to increase hydrophilicity by activating cell attachment via protein molecules, respectively. Modifications with TCP and gelatin enhanced the compression modulus by about 70%, and attachment of Saos-2 cells by 60%, respectively. Release of the antibiotic demonstrated effective antimicrobial activity against E. coli. The bioactive scaffolds were shown to be good candidates for bone tissue engineering applications.     

Novel capillary channel fiber scaffolds for guided tissue engineering

A novel type of capillary channel fibers (CCFs) containing eight open grooves with depth of 5–15 μm and width of 10 μm were tested for their use in tissue engineering as matrices that provide topographical guidance to neo-tissue development. The matrices fabricated from fibers of poly(l-lactic acid) (PLA) and polyethylene terephthalate (PET) were seeded with rat skin fibroblasts (RSFs) and rat aortic smooth muscle cells (RASMCs) for up to 4 weeks. Cells attached and extended their cytoplasmic lamellapodia within the grooves. The cells were proliferating within the grooves and were highly aligned parallel to the direction of the grooves. RASMCs and RSFs showed highly aligned actin and vimentin cytoskeleton, respectively. The cells also deposited extracellular matrix proteins such as laminin and collagen within the grooves parallel to the groove direction. These CCFs also have the unique ability to move fluids instantaneously by capillary action, thus, have the potential to transport oxygen and nutrients deep within the scaffolds. Such CCF matrices would be useful for creating highly organized tissues such as tendon, ligament, nerve, and cardiac muscle.     

Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering

Bionanocomposites formed by combining biodegradable polymers and nanosized osteoconductive inorganic solids have been regarded as promising biomimetic systems which possess much improved structural and functional properties for bone tissue regeneration. In this study three-dimensional nanocomposite scaffolds based on calcium phosphate (Ca-P)/poly(hydroxybutyrate–co-hydroxyvalerate) (PHBV) and carbonated hydroxyapatite (CHAp)/poly(l-lactic acid) (PLLA) nanocomposite microspheres were successfully fabricated using selective laser sintering, which is a rapid prototyping technology. The sintered scaffolds had controlled material microstructure, totally interconnected porous structure and high porosity. The morphology and mechanical properties of Ca-P/PHBV and CHAp/PLLA nanocomposite scaffolds as well as PHBV and PLLA polymer scaffolds were studied. In vitro biological evaluation showed that SaOS-2 cells had high cell viability and normal morphology and phenotype after 3 and 7 days culture on all scaffolds. The incorporation of Ca-P nanoparticles significantly improved cell proliferation and alkaline phosphatase activity for Ca-P/PHBV scaffolds, whereas CHAp/PLLA nanocomposite scaffolds exhibited a similar level of cell response compared with PLLA polymer scaffolds. The nanocomposite scaffolds provide a biomimetic environment for osteoblastic cell attachment, proliferation and differentiation and have great potential for bone tissue engineering applications.     

Novel porous aortic elastin and collagen scaffolds for tissue engineering

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.     

Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering

Polycaprolactone (PCL) is a bioresorbable polymer with potential applications for bone and cartilage repair. In this work, porous PCL scaffolds were computationally designed and then fabricated via selective laser sintering (SLS), a rapid prototyping technique. The microstructure and mechanical properties of the fabricated scaffolds were assessed and compared to the designed porous architectures and computationally predicted properties. Scaffolds were then seeded with bone morphogenetic protein-7 (BMP-7) transduced fibroblasts and implanted subcutaneously to evaluate biological properties and to demonstrate tissue in-growth. The work done illustrates the ability to design and fabricate PCL scaffolds with porous architecture that have sufficient mechanical properties for bone tissue engineering applications using SLS. Compressive modulus and yield strength values ranged from 52 to 67 MPa and 2.0 to 3.2 Mpa, respectively, lying within the lower range of properties reported for human trabecular bone. Finite element analysis (FEA) results showed that mechanical properties of scaffold designs and of fabricated scaffolds can be computationally predicted. Histological evaluation and micro-computed tomography (microCT) analysis of implanted scaffolds showed that bone can be generated in vivo. Finally, to demonstrate the clinical application of this technology, we designed and fabricated a prototype mandibular condyle scaffold based on an actual pig condyle. The integration of scaffold computational design and free-form fabrication techniques presented here could prove highly useful for the construction of scaffolds that have anatomy specific exterior architecture derived from patient CT or MRI data and an interior porous architecture derived from computational design optimization.     

几丁聚糖在组织工程中的应用

支架材料作为组织工程的生物学植入替代物,对细胞移植与引导新组织生长有重要的作用.几丁聚糖可制成无毒性、无刺激性、生物相容性和生物可降解性良好的生物医用材料,在人工皮肤、骨修复材料、手术缝线等方面已广泛应用.本文分析了纯几丁聚糖支架结构和它与其他天然或合成材料复合后的支架结构的物理、化学性质及其独特的生物学功能,同时还进一步介绍了其应用的范例并探讨了发展前景.     

Biodegradable porous scaffolds for tissue engineering

 Three-dimensional scaffolds play an important role in tissue engineering as an adhesive substrate for implanted cells and a physical support to guide the formation of new organs. The scaffolds should facilitate cell adhesion, promote cell growth, allow the retention of differentiated cell functions, and be biocompatible, biodegradable, highly porous with a large surface-to-volume ratio, mechanically strong, and malleable. A number of biodegradable three-dimensional scaffolds have been developed for tissue engineering. This paper reviews some of the recent events in the development of these scaffolds. Received: March 6, 2002     

Bilayered scaffolds for osteochondral tissue engineering

Osteoarthritis (OA) is a prevalent degenerative joint disease that places a significant burden on the socioeconomic efficacy of communities around the world. Tissue engineering repair of articular cartilage in synovial joints represents a potential OA treatment strategy superior to current surgical techniques. In particular, osteochondral tissue engineering, which promotes the simultaneous regeneration of articular cartilage and underlining subchondral bone, may be a clinically relevant approach toward impeding OA progression. The unique and complex functional demands of the two contrasting tissues that comprise osteochondral tissue require the use of bilayered scaffolds to promote individual growth of both on a single integrated implant. This paper reviews the three current bilayered scaffold strategies applied to solve this challenging problem, with a focus on the need for an innovative approach to design and fabrication of new optimized scaffold combinations to reinforce materials science as an important element of osteochondral tissue engineering.     

牙髓牙本质组织工程生物支架材料的研究进展

背景：组织工程方法中选择合适的支架是关键性的步骤。 目的：回顾分析牙髓牙本质组织工程中支架材料的应用研究。 方法：由第一作者检索1993至2012年 PubMed数据及万方数据库有关牙髓牙本质组织工程中支架材料应用研究等方面的文献。 结果与结论：在牙髓牙本质组织工程中有包括天然生物、人工合成材料和复合材料在内的大量生物材料可供选择,每一种材料都有各自的生物学特点。其中胶原、聚酯、羟基磷灰石等是研究较多的支架材料。自组装多肽水凝胶是由氨基酸制成的新型支架材料,满足理想牙髓牙本质组织工程支架材料的大部分要求,是一种前景广阔的牙髓牙本质组织工程支架材料。     

Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: investigation for cartilage tissue engineering

Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage.     

Jellyfish collagen scaffolds for cartilage tissue engineering

Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans.     

Multilayered silk scaffolds for meniscus tissue engineering

Removal of injured/damaged meniscus, a vital fibrocartilaginous load-bearing tissue, impairs normal knee function and predisposes patients to osteoarthritis. Meniscus tissue engineering solution is one option to improve outcomes and relieve pain. In an attempt to fabricate knee meniscus grafts three layered wedge shaped silk meniscal scaffold system was engineered to mimic native meniscus architecture. The scaffolds were seeded with human fibroblasts (outside) and chondrocytes (inside) in a spatial separated mode similar to native tissue, in order to generate meniscus-like tissue in vitro. In chondrogenic culture in the presence of TGF-b3, cell-seeded constructs increased in cellularity and extracellular matrix (ECM) content. Histology and Immunohistochemistry confirmed maintenance of chondrocytic phenotype with higher levels of sulfated glycosaminoglycans (sGAG) and collagen types I and II. Improved scaffold mechanical properties along with ECM alignment with time in culture suggest this multiporous silk construct as a useful micro-patterned template for directed tissue growth with respect to form and function of meniscus-like tissue.     

Biomimetic nanofibrous scaffolds for bone tissue engineering

Bone tissue engineering is a highly interdisciplinary field that seeks to tackle the most challenging bone-related clinical issues. The major components of bone tissue engineering are the scaffold, cells, and growth factors. This review will focus on the scaffold and recent advancements in developing scaffolds that can mimic the natural extracellular matrix of bone. Specifically, these novel scaffolds mirror the nanofibrous collagen network that comprises the majority of the non-mineral portion of bone matrix. Using two main fabrication techniques, electrospinning and thermally-induced phase separation, and incorporating bone-like minerals, such as hydroxyapatite, composite nanofibrous scaffolds can improve cell adhesion, stem cell differentiation, and tissue formation. This review will cover the two main processing techniques and how they are being applied to fabricate scaffolds for bone tissue engineering. It will then cover how these scaffolds can enhance the osteogenic capabilities of a variety of cell types and survey the ability of the constructs to support the growth of clinically relevant bone tissue.     

Electrospun nanostructured scaffolds for bone tissue engineering

The current challenge in bone tissue engineering is to fabricate a bioartificial bone graft mimicking the extracellular matrix (ECM) with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Biocomposite polymeric nanofibers containing nanohydroxyapatite (HA) fabricated by electrospinning could be promising scaffolds for bone tissue engineering. Nanofibrous scaffolds of poly-l-lactide (PLLA, 860 ± 110 nm), PLLA/HA (845 ± 140 nm) and PLLA/collagen/HA (310 ± 125 nm) were fabricated, and the morphology, chemical and mechanical characterization of the nanofibers were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy and tensile testing, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was also assessed by growing human fetal osteoblasts (hFOB), and investigating the proliferation, alkaline phosphatase activity (ALP) and mineralization of cells on different nanofibrous scaffolds. Osteoblasts were found to adhere and grow actively on PLLA/collagen/HA nanofibers with enhanced mineral deposition of 57% higher than the PLLA/HA nanofibers. The synergistic effect of the presence of an ECM protein, collagen and HA in PLLA/collagen/HA nanofibers provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation. The results of our study showed that the biocomposite PLLA/collagen/HA nanofibrous scaffold could be a potential substrate for the proliferation and mineralization of osteoblasts, enhancing bone regeneration.     

Biomaterials and scaffolds for ligament tissue engineering

Tissue engineering has achieved much progress in an attempt to improve and recover impaired functions of tissues and organs. Although many studies have been done, progress for tissue-engineered anterior cruciate ligaments (ACLs) has been slow due to their complex structures and mechanical properties. In this review, the ACL anatomical structure, progresses achieved, material selection, structure design, and future direction have been discussed, while the challenges and requirements from materials and scaffolds are highlighted. There is a considerably huge amount work that needs to be carried out; as such, future direction in ligament tissue engineering is proposed in hope that this review will give information on future ligament tissue engineering.