Beneficial effect of hydrophilized porous polymer scaffolds in tissue-engineered cartilage formation

Three dimensional (3D) porous poly(L-lactic acid) (PLLA) scaffolds were fabricated using a modified gas foaming method whose effervescent porogens were a mixture of sodium bicarbonate and citric acid. To improve chondrocyte adhesion, the scaffolds were then hydrophilized through oxygen plasma treatment and in situ graft polymerization of acrylic acid (AA). When the physical properties of AA-grafted scaffolds were examined, the porosity and pore size were 87 approximately 93% and 100 approximately 300 microm, respectively. The pore sizes were highly dependent on the varying ratios (w/w) between porogen and polymer solution. Influenced by their pore sizes, the compressive moduli of scaffolds significantly decreased with increasing pore size. The altered surface characteristics were clearly reflected in the reduced water contact angles that meant a significant hydrophilization with the modified polymer surface. Electron spectroscopy for chemical analysis (ESCA) and time-of-flight secondary ion mass spectrometer (ToF-SIMS) also confirmed the altered surface chemistry. When chondrocytes were seeded onto the AA-grafted PLLA scaffolds, cell adhesion and proliferation were substantially improved as compared to the unmodified scaffolds. The benefit of the modified scaffolds was clear in the gene expressions of collagen type II that was significantly upregulated after 4-week culture. Safranin-O staining also identified greater glycosaminoglycan (GAG) deposition in the modified scaffold. The AA-grafted porous polymer scaffolds were effective for cell adhesion and differentiation, making them a suitable platform for tissue-engineered cartilage.     

制备软骨组织工程支架的方法

背景：软骨组织工程支架作为软骨细胞外基质的替代物,其外形和孔结构对实现其作用和功能具有非常重要的意义。 目的：回顾目前若干种常用软骨组织工程中三维多孔支架的制备方法。 方法：由第一作者检索2000至2013年PubMed数据库,ELSEVIER SCIENCEDIRECT、万方数据库、中国知网数据库。英文检索词为“Cartilage tissue engineering;scaffolds;fabrication”,中文检索词为“软骨组织工程;制备方法;支架材料;多孔支架”。 结果与结论：制备软骨组织工程支架的方法有相分离/冷冻干燥法、水凝胶技术、快速成型技术、静电纺丝法、溶剂浇铸/粒子沥滤法及气体发泡法等。目前研究发现,支架中孔径的大小对组织的重建有着直接的影响,孔径为100-250 μm的孔有益于骨及软骨组织的再生。通过溶液浇铸/粒子沥滤法、气体发泡法所制备的支架孔径大小在这一范围内,因此比较适合用于骨、软骨组织工程支架的构建。研究人员通常将多种方法结合起来,以期能制备出生物和力学性能方面更加仿生的组织工程多孔支架。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Hyalograft C: hyaluronan-based scaffolds in tissue-engineered cartilage

Articular cartilage injuries have poor reparative capability and, if left untreated, may progress to osteo-arthritis. Unsatisfactory results with conventional treatment methods have prompted the development of innovative solutions including the use of cell transplantations, with or without a supporting scaffold. Tissue engineering combines cells, scaffolds and bio-active factors, which represents one of the most promising approaches for the restoration of damaged tissues. Available today, hyaluronan, also known as hyaluronic acid, is a natural glycosaminoglycan present in all soft tissues of higher organisms and in particularly high concentrations in the extracellular matrix of articular cartilage and in the mesenchyme during embryonic development in which it plays a number of biological functions, not only as a structural component but as an informational molecule as well. Moreover, hyaluronan can be manufactured in a variety of physical forms including hydrogels, sponges, fibres and fabrics allowing to develop a variety of hyaluronan-based scaffolds. This review will present both theoretical and experimental evidences that led to the development of Hyalograft C, an exploitation of hyaluronic acid technology and a tissue engineering approach for the resolution of articular cartilage defects.     

组织工程软骨构建中不同支架材料的特征

BACKGROUND: Most scholars believe that the cartilage tissue engineering is a new direction for the treatment of cartilage defects. It has made partial progress in the basic research, and to construct a scaffold is essential in cartilage tissue engineering. OBJECTIVE: To systematically review and summarize the application of different tissue-engineered scaffolds in articular cartilage repair. METHODS: A computer-based search of CNKI, VIP and PubMed databases was performed for relevant basic research literatures published from January 2005 to January 2015 using the keywords of “tissue engineering,  scaffold, cartilage” in Chinese and English, respectively. Meanwhile, references in the retrieved articles were retrieved manually. RESULTS AND CONCLUSION: For selection and preparation of tissue-engineered cartilage scaffolds, it is necessary to fully take into account the advantages and disadvantages of natural hydrogel materials, synthetic scaffolds, composite scaffolds, nano-scaffolds, and injectable scaffolds. Currently, there is still no normal transition between all tissue-engineered cartilage scaffolds and the natural cartilage. Therefore, to develop nano-multilayer integrated scaffolds with the calcified cartilage layer is expected to become one of the research focuses of bone and cartilage tissue engineering.      

制备软骨组织工程支架的材料和方法

背景：软骨组织工程的研究为修复软骨缺损提供了新的思路和方法,其中如何获得理想的组织工程支架是这一研究的核心和难点。 目的：回顾性分析软骨组织工程支架的材料选择和制备方法。 方法：由第一作者检索2000至2012年 PubMed数据库、ELSEVIER SCIENCEDIRECT、万方数据库、中国知网库有关制备软骨组织工程支架的材料选择和方法等方面的文献。 结果与结论：软骨支架材料分为天然生物材料、人工合成高分子材料和复合材料。可采用相分离法、溶剂浇铸/粒子沥滤技术、气体发泡技术、快速成型技术及静电纺丝法制备支架材料。由于胶原、琼脂糖和藻酸盐等水凝胶类天然材料可提供足够的生物相容性、增殖和黏附能力及亲水性,电纺的人工合成高分子材料复合支架又可以保证支架的力学强度、塑形要求、孔隙率、可降解性等,将天然材料利用包埋技术和表面修饰技术复合于电纺的高分子复合材料支架上将更有利于支架性能的发挥。     

Evaluation of type II collagen scaffolds reinforced by poly(epsilon-caprolactone) as tissue-engineered trachea

A novel composite scaffold comprising a poly(epsilon-caprolactone) (PCL) stent and a type II collagen sponge for tissue-engineered trachea was developed. The PCL stent with surface grooves was fabricated by casting and freeze drying the PCL solution in a mold container. The grooves on the stent were filled by the type II collagen with crosslinking treatment (ring-shaped collagen sponge). The rabbit chondrocytes (3 x 10(6) cells for each ring) were seeded onto the collagen sponge of the scaffold. The cell-scaffold constructs were implanted subcutaneously in the dorsum of nude mice. After 4 and 8 weeks, constructs were harvested and dedicated for measurement of mechanical properties, histology, and biochemical assays. It was found that the constructs were strong enough to retain their tubular shape against extrinsic forces in the dorsum of nude mice. The gross appearance of the constructs revealed cartilage-like tissue at 8 weeks, with modulus higher than that of native trachea. Histological and biochemical analyses of the tissue-engineered tracheal cartilage revealed evenly spaced lacunae embedded in the matrix, with abundant proteoglycans and type II collagen. The stent-sponge composite facilitated the proliferation of chondrocytes and was expected to provide adequate mechanical strength, and therefore was a promising material for use in trachea tissue engineering.     

Pore size effect of collagen scaffolds on cartilage regeneration

Scaffold pore size is an important factor affecting tissue regeneration efficiency. The effect of pore size on cartilage tissue regeneration was compared by using four types of collagen porous scaffolds with different pore sizes. The collagen porous scaffolds were prepared by using pre-prepared ice particulates that had diameters of 150–250, 250–355, 355–425 and 425–500 μm. All the scaffolds had spherical large pores with good interconnectivity and high porosity that facilitated cell seeding and spatial cell distribution. Chondrocytes adhered to the walls of the spherical pores and showed a homogeneous distribution throughout the scaffolds. The in vivo implantation results indicated that the pore size did not exhibit any obvious effect on cell proliferation but exhibited different effects on cartilage regeneration. The collagen porous scaffolds prepared with ice particulates 150–250 μm in size best promoted the expression and production of type II collagen and aggrecan, increasing the formation and the mechanical properties of the cartilage.     

Fluid flow increases type II collagen deposition and tensile mechanical properties in bioreactor-grown tissue-engineered cartilage

A novel parallel-plate bioreactor has been designed to apply a consistent level of fluid flow-induced shear stress to tissue-engineered articular cartilage in order to improve the matrix composition and mechanical properties and more nearly approximate to that of native tissue. Primary bovine articular chondrocytes were seeded into the bioreactor at high densities (1.7 x 10(6) cell/cm2) without a scaffold and cultured for two weeks under static, no-flow conditions. A mean fluid flow-induced shear stress of 1 dyne/cm2 was then applied continuously for 3 days. The application of flow produced constructs with significantly (p < 0.05) higher amounts of total collagen (via hydroxyproline) and specifically type II collagen (via ELISA) (25.3 +/- 2.5% and 22.1 +/- 4.7% of native tissue, respectively) compared to static controls (22.4 +/- 1.7% and 9.5 +/- 2.3%, respectively). Concurrently, the tensile Young's modulus and ultimate strength were significantly increased in flow samples (2.28 +/- 0.19 MPa and 0.81 +/- 0.07 MPa, respectively) compared to static controls (1.55 +/- 0.10 MPa and 0.62 +/- 0.05 MPa, respectively). This study suggests that flow-induced shear stresses and/or enhanced mass transport associated with the hydrodynamic environment of our novel bioreactor may be an effective functional tissue-engineering strategy for improving matrix composition and mechanical properties in vitro.     

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.     

Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer.

The purpose of this study was to determine if chondrocytes cultured through several subcultures at very low plating density would produce new cartilage matrix after being reimplanted in vivo with or without a hydrogel polymer scaffold. Chondrocytes were initially plated in low-density monolayer culture, grown to confluence, and passaged four times. After each passage cells were suspended in purified porcine fibrinogen and injected into the subcutaneous space of nude mice while simultaneously polymerizing with thrombin to reach a final concentration of 40 million cells/cc. Controls were made by injecting fresh, uncultured cells with fibrin polymer and by injecting the cultured cells in saline (without polymer). All samples were harvested at 6 weeks. When injected in polymer, both fresh cells and cells that had undergone only one passage in culture produced cartilaginous nodules. Cultured cells did not produce cartilage, regardless of length of time spent in culture, when injected without polymer. Cartilage was also not recovered from samples with cells kept in culture for longer than one passage, even when provided with a polymer matrix. All samples harvested were subjected to histological analysis and assayed for total DNA, glycosaminoglycan (GAG), and type II collagen. There was histological evidence of cartilage in the groups that used fresh cells and cultured cells suspended in fibrin polymer that only underwent one passage. No other group contained areas that would be consistent with cartilage histologically. All experimental samples had a higher percent of DNA than native swine cartilage, and there was no statistical difference between the DNA content of the groups containing cultured or fresh cells in fibrin polymer. Whereas the GAG content of native cartilage was 8.39% of dry weight and fresh cells in fibrin polymer was 12.85%, cultured cells in fibrin polymer never exceded the 2.48% noted from first passage cells. In conclusion, this study demonstrates that porcine chondrocytes that have been cultured in monolayer for one passage will produce cartilage in vivo when suspended in fibrin polymer.     

Localization of type I and II collagen during development of human first rib cartilage

The localization of fibrillar type I and II collagen was investigated by immunofluorescence staining with specific antibodies in order to obtain a better understanding of tissue remodelling during the development of first rib cartilage. In childhood and early adolescence type I collagen was found to be restricted to the perichondrium of first rib cartilage, while type II collagen was localized in the matrix of hyaline cartilage. However, in advanced age type I collagen was also found in the territorial matrix of intermediate and central chondrocytes of first rib cartilage. The matrix of subperichondrial chondrocytes was negative for type I collagen. This suggests that some chondrocytes in first rib cartilage undergo a modulation to type I collagen-producing cells. The first bone formation was observed in rib cartilages of 20- to 25-year-old adults. Interestingly, the ossification began peripherally, adjacent to the innermost layer of the perichondrium where areas of fibrocartilage had developed. The newly formed bone matrix showed strong immunostaining for type I collagen. Fibrocartilage bordering peripherally on bone matrix revealed only a faint staining for type I collagen, but strong immunoreactivity to type II collagen. The interterritorial matrix of the central chondrocytes failed to react with the type II collagen antibody, in both men and women, from the end of the second decade. These observations indicate that major matrix changes occur at the same time in male and female first rib cartilages. Thus, our findings indicate that ossification in human first rib cartilage does not follow the same pattern as that observed in endochondral ossification of epiphyseal discs or sternal cartilage.     

Construction of tissue-engineered urinary bladder using biodegradable polymer matrices as cell scaffolds

INTRODUCTION　　 Approximately 4 0 0 million personsworldwide suffer from bladder disease. Indi-viduals with end-stage bladder disease often require bladder replacement or repair.Several bladdersubstituteshavebeen attempted with both organicmaterialsand syn-thetics.These attempts have usually failed due to mechanical,structural,or bio-compatibility problems.Segments of autologous stomach and bowel are commonlyused as tissuesforbladderreplacementorrepair,buthave been associated with mul-ti…     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissueengineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

Covalent interactions of type IX collagen in cartilage

The cross-linking of type IX collagen in fetal bovine cartilage was investigated. The main cross-link was dihydroxy-lysinonorleucine (borohydride-reduced) with a lesser amount of the mature cross-link, pyridinoline. Dihydroxylysinonorleucine was present in all three chains of the COL2 domain of the type IX molecule, but only two of them contained pyridinoline even in mature cartilage. Amino acid sequence analysis of individual tryptic peptides that contained 3H-labeled cross-links showed that they had derived from sites of covalent interaction between type IX collagen and the telopeptide sequences of type II collagen. One two-chained peptide was a helical sequence of alpha 2 (IX) COL2 linked to an alpha 1 (II) N-telopeptide. A second peptide was a different helical sequence from another type IX chain linked to an alpha 1(II) c-telopeptide. This latter helical sequence was also the principal site of pyridinoline cross-linking in type IX collagen.     

Influence of cartilage proteoglycans on type II collagen fibrillogenesis

The effects of various proteoglycan samples, isolated from human articular cartilage of different ages, on the rate of the lateral growth phase of the fibril formation of collagen type II were studied by turbidimetry. In general, proteoglycan aggregates accelerate fibrillogenesis, whereas non-aggregating proteoglycans retard this process. The only exception were non-aggregating proteoglycans from very young cartilage, which stimulated the fibril formation strongly. The extent of stimulation by proteoglycans from hip and knee cartilage were compared. The effects of non-aggregating proteoglycans dominate those of aggregated proteoglycans. Chondroitinase ABC digestion of proteoglycan samples did not change the effects on the fibrillogenesis of collagen type II, when these samples were isolated from 18 years-old knee cartilage. The collagen fibril formation was less stimulated in the presence of ABC-ase digested proteoglycan samples from 0-3 month-old knee cartilage, suggesting a primary role for keratan sulphate and a possible influence of chondroitin sulphate when keratan sulphate is not present. Only proteoglycans from very old cartilage were able to reduce the amount of collagen fibrils formed in vitro. Proteoglycans could not be detected bound to the fibril pellet despite the fact that part of the pellet was not dissolvable in acetic acid. It is concluded that proteoglycans may play a regulatory role in collagen type II fibril formation in articular cartilage.     

Short-term human chondrocyte culturing on oriented collagen coated gelatine scaffolds for cartilage replacement

The biological, biochemical, mechanical, and structural properties of artificial scaffolds for tissue engineering are known to be of great importance. Therefore, in this study a hydrogel derived scaffold with biomechanical and structural properties similar to native articular cartilage was synthesized. The gelatine-based hydrogel was processed by freeze-structuring, structuring by electrochemical water-decomposition, freeze-drying and chemical fixation resulting in a defined scaffold-structure. By electron microscopy a perpendicular pore-channel structure was verified with channel diameters between 30 microm and 70 microm. Mechanical testing showed mechanical properties similar to native cartilage. Human chondrocytes from biopsy samples were cultivated on these hydrogel scaffolds for three days. Two different cell densities (1.2 x 10(6) cells/cm3 and 12.0 x 10(6) cells/cm3) were used for cultivation. Histology of the cell seeded artificial scaffolds demonstrated vital cells that are widely distributed within the scaffold and mimic a columnar arrangement.     

Effects of acid type on physical and biological properties of collagen scaffolds

Different acid types, hydrochloric acid and acetic acid, were used to prepare collagen solutions. The effects of acid types on physical and biological properties of collagen scaffolds were investigated. The collagen solution prepared with hydrochloric acid (C-HCl) was much more viscous than that prepared with acetic acid (C-Acetic). The conformation of collagen chains was expected to be different due to differences in ionic strength of collagen solutions. Morphology of the scaffolds analyzed by scanning electron microscopy (SEM) revealed that C-HCl scaffolds had larger pore sizes (around 100 microm) than C-Acetic scaffolds (around 50 microm). The more viscous C-HCl solution resulted in harder scaffolds shown by higher compressive modulus. However, the swelling ability of the C-Acetic scaffolds was higher in phosphate-buffered saline solution. The results from in vitro L929 cell culture showed that C-HCl scaffolds could promote cell initial attachment while the C-Acetic scaffolds enhanced cell proliferation. Therefore, the type of acid used as a solvent to form collagen solution affects the morphology, physical and biological properties of the resulting collagen scaffolds.     

Characterization of tissue-engineered scaffolds microfabricated with PAM

PAM (pressure-activated microsyringe) is a new microfabrication technique that allows the fabrication of two- and three-dimensional scaffolds with a well-defined geometry using polymers soluble in volatile solvents. In this study, polymeric scaffolds were realized with four different polymers--PCL, PLLA, PLGA, and a blend of PCL and PLLA--and with three different geometries-square grids, hexagonal grids, and octagonal grids. The scaffolds were characterized in terms of porosity, hydrophilicity, cell adhesion, and their mechanical properties. An analysis of the measured data shows that the physical and mechanical properties of the scaffold depend on its geometry and line width, both of which are easily modulated using PAM.     

胶原-壳聚糖制备仿生多层结构软骨支架

目的制备结构与天然软骨结构相似的仿生多层软骨支架。方法采用先后于-20℃和液氮中冷冻的预冻方式,冷冻干燥法制备双层支架。采用-20℃冷冻后,部分熔融再液氮重冻的预冻方式,冷冻干燥制备了厚度约2mm的仿生多层软骨支架。采用XRD和红外光谱观察胶原和壳聚糖的复合情况。采用SEM观察支架的形貌。对比了纯壳聚糖支架、纯胶原支架、胶原壳聚糖复合材料单层支架和仿生多层支架在干燥和湿润两种状态下的力学性能。结果胶原和壳聚糖的复合存在化学反应,复合材料形成更好的孔结构,仿生多层支架从上至下分别具有致密层结构,圆形孔结构和垂直孔结构。支架材料在干燥和湿润状态下的力学性能有很大差别,仿生多层支架在湿润状态下各层具有不同的力学性能。结论仿生多层软骨支架的结构接近于天然关节软骨多层结构,且在湿润状态下各层的力学性能有差异,有望更好地维持软骨细胞表型和提高软骨损伤修复效果。