Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds

Chitosan has been shown to be a promising scaffold for various applications in tissue engineering. In this study, a chitosan-gelatin complex was fabricated as a scaffold by a freezing and lyophilizing technique. Chitosan's structure and characteristics are similar to those of glycosaminoglycan (GAG) and its analogs, and possesses various biological activities, whereas gelatin can serve as a substrate for cell adhesion, differentiation, and proliferation. With the use of autologous chondrocytes isolated from pig's auricular cartilage and seeded onto the chitosan-gelatin scaffold, elastic cartilages have been successfully engineered at the porcine abdomen subcutaneous tissue. After 16 weeks of implantation, the engineered elastic cartilages have acquired not only normal histological and biochemical, but also mechanical properties. The tissue sections of the engineered elastic cartilages showed that the chondrocytes were enclosed in the lacuna, similar to that of native cartilage. The presence of elastic fibers in the engineered cartilages was also demonstrated by Vehoeff's staining, and immunohistochemical staining confirmed the presence of type II collagen in the engineered cartilages. Quantitatively, the GAG in the engineered cartilages reached 90% of the concentration in native auricular cartilage. Furthermore, biomechanical analysis demonstrated that the extrinsic stiffness of the engineered cartilages reached 85% of the level in native auricular cartilage when it was harvested at 16 weeks. Thus, this study demonstrated that the chitosan-gelatin complex may serve as a suitable scaffold for cartilage tissue engineering.     

Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study

Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue. Biphasic implants consisting of pre-fabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements. Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro. Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-L-lactide [P(L)LA], poly-d,l-lactide [P(D,L)LA] or Collagen-hydroxyapatite [Col-HA] and were cultured for 15 weeks. Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation. P(D,L)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape. Little or no breakdown or chondrocyte colonisation was evident in P(L)LA. Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial. These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.     

Tissue engineering of bovine articular cartilage within porous poly(ether ester) copolymer scaffolds with different structures

The potential of porous poly(ether ester) scaffolds made from poly(ethylene glycol) terephthalate: poly(butylene terephthalate) (PEGT:PBT) block copolymers produced by various methods to enable cartilaginous tissue formation in vitro was studied. Scaffolds were fabricated by two different processes: paraffin templating (PT) and compression molding (CM). To determine whether PEGT:PBT scaffolds are able to support chondrogenesis, primary bovine chondrocytes were seeded within cylindrical scaffolds under dynamic seeding conditions. On day 3, constructs were transferred to six-well plates and evaluated for glycosaminoglycan (GAG) distribution (3, 10, and 24 days), type II collagen distribution, cellularity, and total collagen and GAG content (10 and 24 days). It was observed that better cell distribution during infiltration within PT scaffolds allowed greater chondrogenesis, and at later time points, than in CM scaffolds. The amount of GAG remained constant for all groups from 10 to 24 days, whereas collagen content increased significantly. These data suggest that PEGT:PBT scaffolds are suitable for cartilage tissue engineering, with the PT process enabling greater chondrogenesis than CM.     

Tissue engineering of articular cartilage using an allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold (ACHMS scaffold)

The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or non-filled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects.     

Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: an animal model

Biodegradable macroporous cryogels with highly open and interconnected pore structures were produced from dextran modified with oligo L-lactide bearing hydroxyethylmethacrylate (HEMA) end groups in moderately frozen solutions. Tissue responses to these novel scaffolds were evaluated in rats after dorsal subcutaneous implantation, iliac submuscular implantation, auricular implantation, or in calvarial defect model. In no case, either necrosis or foreign body reaction was observed during histological studies. The cryogel scaffolds integrated with the surrounding tissue and the formation of a new tissue were accompanied with significant ingrowth of connective tissue cells and new blood vessels into the cryogel. The tissue responses were significantly lower in auricular and calvarial implantations when compared with the subcutanous and the submuscular implantations. The degradation of the scaffold was slower in bone comparing to soft tissues. The biodegradable cryogels are highly biocompatible and combine extraordinary properties including having soft and elastic nature, open porous structure, and very rapid and controllable swelling. Therefore, the cryogels could be promising candidates for further clinical applications in tissue regeneration.     

Poly(dopamine) coating of scaffolds for articular cartilage tissue engineering

A surface modification technique based on poly(dopamine) deposition developed from oxidative polymerization of dopamine is known to promote cell adhesion to several cell-resistant substrates. In this study this technique was applied to articular cartilage tissue engineering. The adhesion and proliferation of rabbit chondrocytes were evaluated on poly(dopamine)-coated polymer films, such as polycaprolactone, poly(L-lactide), poly(lactic-co-glycolic acid) and polyurethane, biodegradable polymers that are commonly used in tissue engineering. Cell adhesion was significantly increased by merely 15 s of dopamine incubation, and 4 min incubation was enough to reach maximal cell adhesion, a 1.35-2.69-fold increase compared with that on the untreated substrates. Cells also grew much faster on the poly(dopamine)-coated substrates than on untreated substrates. The increase in cell affinity for poly(dopamine)-coated substrates was demonstrated via enhancement of the immobilization of serum adhesive proteins such as fibronectin. When the poly(dopamine)-coating technique was applied to three-dimensional (3-D) polyurethane scaffolds, the proliferation of chondrocytes and the secretion of glycosaminoglycans were increased compared with untreated scaffolds. Our results show that the deposition of a poly(dopamine) layer on 3-D porous scaffolds is a simple and promising strategy for articular cartilage tissue engineering, and may be applied to other types of tissue engineering.     

工程软骨支架的临床应用研究进展

移植工程软骨修复软骨损伤是目前较为理想的治疗方法,构建工程软骨需要种子细胞和支架材料,支架材料的性能对工程软骨的生物特性有重要影响.讨论支架材料的研究进展,比较不同支架材料的工程软骨的临床应用结果,对进一步改善工程软骨的生物学性能有重要意义.结合近年来工程软骨支架的研究和临床应用情况作一综述和展望.     

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.     

Tissue engineering bone-ligament complexes using fiber-guiding scaffolds

Regeneration of bone-ligament complexes destroyed due to disease or injury is a clinical challenge due to complex topologies and tissue integration required for functional restoration. Attempts to reconstruct soft-hard tissue interfaces have met with limited clinical success. In this investigation, we manufactured biomimetic fiber-guiding scaffolds using solid free-form fabrication methods that custom fit complex anatomical defects to guide functionally-oriented ligamentous fibers in vivo. Compared to traditional, amorphous or random-porous polymeric scaffolds, the use of perpendicularly oriented micro-channels provides better guidance for cellular processes anchoring ligaments between two distinct mineralized structures. These structures withstood biomechanical loading to restore large osseous defects. Cell transplantation using hybrid scaffolding constructs with guidance channels resulted in predictable oriented fiber architecture, greater control of tissue infiltration, and better organization of ligament interface than random scaffold architectures. These findings demonstrate that fiber-guiding scaffolds drive neogenesis of triphasic bone-ligament integration for a variety of clinical scenarios.     

Novel poly(ethylene glycol) scaffolds crosslinked by hydrolyzable polyrotaxane for cartilage tissue engineering

Highly porous poly(ethylene glycol) (PEG) hydrogel scaffolds crosslinked with hydrolyzable polyrotaxane for cartilage tissue engineering were prepared by a solvent casting/salt leaching technique. The resultant scaffolds have well interconnected microporous structures ranging from 87 to 90%. Pore sizes ranging from 115.5-220.9 microm appeared to be dependent on the size of the sieved sodium chloride particulates. Moreover, a dense surface skin layer was not found on either side of the scaffold surfaces. Using microscopic Alcian blue staining of the chondrocyte-seeded scaffolds, well adhered cells and newly produced glycosaminoglycans (GAG) were confirmed. Following the initial chondrocyte seeding onto the hydrogel scaffolds, the cell number was significantly increased, reaching 149, 877, and 1228 cells/mg of tissue at 8, 15, and 21 days in culture, respectively. The micrograph shows well adhered and spread chondrocytes in the interior pores and a cartilaginous extracellular matrix with a GAG fraction produced from the chondrocytes. Results suggest that the PEG hydrogel scaffolds crosslinked with the hydrolyzable polyrotaxane are a promising candidate for chondrocyte culture.     

Development of arginine-glycine-aspartate-immobilized 3D printed poly(propylene fumarate) scaffolds for cartilage tissue engineering

Abstract

Poly(propylene fumarate) (PPF) has known to be a good candidate material for cartilage tissue regeneration because of its excellent mechanical properties during its degradation processes. Here, we describe the potential application of PPF-based materials as 3D printing bioinks to create macroporous cell scaffolds using micro-stereolithography. To improve cell-matrix interaction of seeded human chondrocytes within the PPF-based 3D scaffolds, we immobilized arginine-glycine-aspartate (RGD) peptide onto the PPF scaffolds. We also evaluated various cellular behaviors of the seeded chondrocytes using MTS assay, microscopic and histological analyses. The results indicated that PPF-based biocompatible scaffolds with immobilized RGD peptide could effectively support initial adhesion and proliferation of human chondrocytes. Such a 3D bio-printable scaffold can offer an opportunity to promote cartilage tissue regeneration.     

Minimally invasive technique of auricular cartilage harvest for tissue engineering

Tissue engineered human cartilage is presently being utilized in clinical research programs in a variety of medical disciplines including otolaryngology, urology, and orthopedics. In this study, we present a new methodology for auricular cartilage harvest that can be applied to tissue engineering. Eight 16-week-old pigs were subjected to a traditional open cartilage harvest technique involving suture closure, while the other ear was subjected to the closed stitchless cartilage harvest, using a 12-gauge core biopsy needle. Surgical time was significantly (p < 0.0001) shorter (3.5 +/- 2.8 min for closed vs. 14.4 +/- 5 min for open), and no sutures where utilized in the closed technique. Sample weights were significantly (p < 0.00001) greater (0.115 +/- 0.028 g vs. 0.045 +/- 0.005 g) for the closed techniques. However, the minimally invasive closed technique had fewer incidents of bruising, hematoma, long-term stitch abscess, and scarring. Cell culture data shows no disadvantage to either technique with regards to cell growth characteristics. Final histological data from donor ears indicates favorable results with the minimally invasive technique. This technique preserves cell viability and isolation efficiency while decreasing surgical time and lessening postoperative complications.     

Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering

Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.     

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

Tissue engineering scaffolds containing embedded fluorinated-zeolite oxygen vectors

Efficient oxygen supply is a continuing challenge for the fabrication of successful tissue engineered constructs with clinical relevance. In an effort to enhance oxygen delivery we report the feasibility of using fluorinated zeolite particles embedded in three-dimensional (3-D) polyurethane scaffolds as novel oxygen vectors. First, 1H,1H,2H,2H-perfluorodecyltriethoxysilane was successfully coupled to zeolite framework particles to examine the dose-dependent dissolved oxygen concentration. Following this, the fluorinated-zeolite (FZ) particles were embedded in 3-D tissue engineering polyurethane scaffolds. Our data demonstrates an even distribution of FZ particles in the 3-D scaffolds without affecting the scaffold porosity or pore size. Human coronary artery smooth muscle cell (HCASMC) proliferation on FZ-containing polyurethane (PCU-FZ) scaffolds was significantly greater than on control scaffolds (P = 0.05). Remarkably, cell infiltration depths on the PCU-FZ scaffolds was double that on PCU control scaffolds. Taken together, our data suggest the potential of PCU-FZ scaffolds for tissue engineering with enhanced oxygen delivery to cells.     

Tissue engineering of biphasic joint cartilage transplants.

In isolated posttraumatic or idiopathic joint defects the chondral layers and adjacent subchondral spongy bone are usually destructed. For regeneration we suggest the in vitro formation of a cartilage-coated biomaterial carriers (biphases) in order to fill the correspondingjoint defects. In this study Biocoral, a natural coralline material made of calcium carbonate, and calcite, a synthetic calcium carbonate, were used as supports for the cultivation of bovine chondrocytes in a three-dimensional polymer fleece. The cell-polymer-structure was affixed to the biomaterial with a fibrin-cell-solution. The artificial cartilage formed a new matrix and fused with the underlying biomaterial. The results indicate a promising technical approach to anchor tissue engineered cartilage in joint defects.     

Biodegradable polymer (D,L-lactide-epsilon-caprolactone) in aortic vascular prosthesis: morphological evaluation in an animal model

Biodegradable D,L-Lactide-epsilon-caprolactone copolymer was used in substitution to bovine collagen to seal porosity in nine Dacron vascular Sorin Carbografts. One served as control and 8 were implanted in mini-pigs as vascular by-pass in the thoracic aorta. The grafts were explanted at 7 days (4 animals), 30 (2 animals) and at 90 days (2 animals), and submitted to gross examination, X-ray, histology and electron microscopy. Aim of the study was to assess the safety and the reliability of these polyester vascular prostheses impregnated with the copolymer in terms of containment of the bleeding in the perioperative period, host inflammatory response, copolymer biodegradation and prostheses "healing" All the grafts were patent at angiographic and X-ray examination. At 7 days blood infiltration between Dacron and copolymer lining was detected. Inflammatory granulocyte infiltrates and granulomatous reaction with polymer degradation was observed at 30 days and fibrous tissue healing at 90 days. Luminal surface was covered by thin thrombi at 7 and by a neointima at 30 and 90 days. We conclude that D,L-Lactide-epsilon-caprolactone copolymer is effective in preventing perigraft bleeding, even though an early hematoma between Dacron and the copolymer coating occurs. Copolymer is degraded through a mild inflammatory reaction, with eventual evolution to fibrous healing.     

Tissue engineering: strategies, stem cells and scaffolds

Tissue engineering scaffolds are designed to influence the physical, chemical and biological environment surrounding a cell population. In this review we focus on our own work and introduce a range of strategies and materials used for tissue engineering, including the sources of cells suitable for tissue engineering: embryonic stem cells, bone marrow-derived mesenchymal stem cells and cord-derived mesenchymal stem cells. Furthermore, we emphasize the developments in custom scaffold design and manufacture, highlighting laser sintering, supercritical carbon dioxide processing, growth factor incorporation and zoning, plasma modification of scaffold surfaces, and novel multi-use temperature-sensitive injectable materials.     

组织工程技术修复损伤关节软骨的研究与应用

背景：软骨是一种无血管的组织,软骨损伤后自身修复能力有限。当前用于治疗关节软骨损伤的方法从保守治疗到手术治疗多种多样。随着组织工程技术的发展,关节软骨的修复又进入了新的高度。 目的：综述组织工程方法修复软骨损伤的新进展。 方法：由第一作者在2013年5月应用计算机检索2000至2013年PubMed 数据库及CNKI 数据库,英文以“cartilage tissue engineering,cartilage defect;stem cell,scaffold;growth factor”为关键词,中文以“软骨组织工程,软骨缺损,干细胞,支架,生长因子”为关键词,选择内容与软骨组织工程、软骨损伤修复相关的文章,同一领域文献则选择近期发表或发表在权威杂志文章,共纳入64篇文献。 结果与结论：软骨组织工程三大要素——种子细胞、支架和细胞因子,三者必须协调发展和互利。现阶段组织工程方法修复关节软骨损伤的研究虽已取得很大进展,但大多停留于实验探索阶段,尚未应用于临床。随着新材料的不断研发,新的组织工程软骨修复材料将兼顾材料学和生物科学的需要,使其更接近机体自身组织生物学特性。在新的技术支持下,动物实验研究也将向临床试验转变,使关节软骨损伤的治疗取得突破性进展。     

Tissue engineering auricular reconstruction: in vitro and in vivo studies

Although investigators have demonstrated that neocartilage can be constituted in a predetermined shape and in complex three-dimensional structures, such as a human ear, by using cell transplantation on polymer constructs, many unsolved problems still remain. The crucial issues for auricular tissue engineering consisted of optimal cell culture environment, choice of polymers, behavior of chondrocytes, study of cell-polymer constructs in an acceptable animal model, and long-term structural integrity. Here we describe our tissue engineering approaches for auricular reconstruction including auricular scaffold fabrication, in vitro chondrogenesis, in vivo immunocompromized xenograft and immunocompetent autologous animal models, and long-term follow-up. Though many current obstacles regarding auricular tissue engineering still exist, we demonstrate techniques of auricular scaffold fabrication with promising in vitro and in vivo neocartilage formation, optimal selection and application of animal models, and, to the best of our knowledge, the first report of different biodegradable biomaterial trials and the longest in vivo results (10 months) for auricular tissue engineering.