Gelatin/chitosan/hyaluronan scaffold integrated with PLGA microspheres for cartilage tissue engineering

Poly(lactide-co-glycotide) (PLGA) microspheres integrated into gelatin/chitosan/hyaluronan scaffolds were fabricated by freeze-drying and crosslinking with 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. The effects of the microspheres on porosity, density, compressive modulus, phosphate-buffered saline uptake ratio and weight loss of the scaffolds were evaluated. Generally, a scaffold with a higher PLGA content had a lower porosity and weight loss, and a medium uptake ratio, but a larger apparent density and compressive modulus. When the PLGA content was lower than 50%, the PLGA-integrated scaffolds had a similar pore size (approximately 200microm) as that of the control, and as much as 90% of their porosity could be preserved. In vitro chondrocyte culture in the 50% PLGA-integrated scaffold demonstrated that the cells could proliferate and secrete extracellular matrix at the same level as in the control gelatin/chitosan/hyaluronan scaffold.     

Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering

Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(l,d-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3 +/- 58.0 microm) rather than smaller (11.15 +/- 11.08 microm) microspheres, generated pores on the order of 200 microm as compared to 20 microm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications.     

Mechanistic studies for monodisperse exenatide-loaded PLGA microspheres prepared by different methods based on SPG membrane emulsification

Poly(DL-lactic-co-glycolic acid) (PLGA) microspheres have been widely prepared by many methods, including solvent evaporation, solvent extraction and the co-solvent method. However, very few studies have compared the properties of microspheres fabricated by these methods. This is partly because the broad size distribution of the resultant particles severely complicates the analysis and affects the reliability of the comparison. To this end, uniform-sized PLGA microspheres have been prepared by Shirasu porous glass premix membrane emulsification and used to encapsulate exenatide, a drug for treating Type 2 diabetes. Based on this technique, the influences on the properties of microspheres fabricated by the aforementioned three methods were intensively investigated, including in vitro release, degradation and pharmacology. We found that these microspheres presented totally different release behaviors in vitro and in vivo, but exhibited a similar trend of PLGA degradation. Moreover, the internal structural evolution visually demonstrated these release behaviors. We selected for further examination the microsphere prepared by solvent evaporation because of its constant release rate, and explored its pharmacodynamics, histology, etc., in more detail. This microsphere when injected once showed equivalent efficacy to that of twice-daily injections of exenatide with no inflammatory response.     

A composite scaffold of PLGA microspheres/fibrin gel for cartilage tissue engineering: fabrication, physical properties, and cell responsiveness

A composite scaffold of poly(L-lactic-co-glycolic acid) (PLGA) microspheres and fibrin gel was fabricated by blending fibrinogen-immobilized PLGA microspheres with fibrinogen and thrombin solution. The PLGA microspheres with a size of 70 approximately 100 microm were aminolyzed in a hexanediamine/n-propanol solution to introduce free amino groups on their surface. The fibrinogen immobilization was achieved by glutaraldehyde coupling. When the --NH(2) content on the microsphere surface was increased from approximately 2 x 10(-8) mol/mg to approximately 4 x 10(-8) mol/mg, the fibrinogen amount was correspondingly increased from approximately 35 microg/mg to approximately 70 microg/mg. Measured by UV-VIS spectroscopy, the clotting time of the composite was less influenced by the microsphere amount, but mainly controlled by the thrombin concentration. When the thrombin concentration was higher than 15 U/mL, the gelation could be finished within 1 min and yielded a composite with evenly suspended and distributed PLGA microspheres. Blending with the microspheres could significantly improve the elastic modulus of the hydrogel as well, whereas less influence on the chondrocyte proliferation and extracellular matrix production.     

Poly(lactide-co-glycolide) microspheres as a moldable scaffold for cartilage tissue engineering

This study demonstrates the use of biodegradable poly(lactide-co-glycolide) (PLG) microspheres as a moldable scaffold for cartilage tissue engineering. Chondrocytes were delivered to a cylindrical mold with or without PLG microspheres and cultured in vitro for up to 8 weeks. Cartilagenous tissue formed using chondrocytes and microspheres maintained thickness, shape, and chondrocyte collagen type II phenotype, as indicated by type II collagen staining. The presence of microspheres further enhanced total tissue mass and the amount of glycosaminoglycan that accumulated. Evaluation of microsphere composition demonstrated effects of polymer molecular weight, end group chemistry, and buffer inclusion on tissue-engineered cartilage growth. Higher molecular weight PLG resulted in a larger mass of cartilage-like tissue formed and a higher content of proteoglycans. Cartilage-like tissue formed using microspheres made from low molecular weight and free carboxylic acid end groups did not display increases in tissue mass, yet a modest increased proteoglycan accumulation was detected. Microspheres comprised of PLG with methyl ester end groups yielded a steady increase in tissue mass, with no real increase in matrix accumulation. The microencapsulation of Mg(OH)(2) had negative effects on tissue mass and matrix accumulation. The data herein reflect the potential utility of a moldable PLG-chondrocyte system for tissue-engineering applications.     

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     

Poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for cartilage tissue engineering

Injectable scaffold has raised great interest for tissue regeneration in vivo, because it allows easy filling of irregularly shaped defects and the implantation of cells through minimally invasive surgical procedures. In this study, we evaluated poly(lactic-co-glycolic acid) (PLGA) microsphere as an injectable scaffold for in vivo cartilage tissue engineering. PLGA microspheres (30-80 microm in diameter) were injectable through various gauges of needles, as the microspheres did not obstruct the needles and microsphere size exclusion was not observed at injection. The culture of chondrocytes on PLGA microspheres in vitro showed that the microspheres were permissive for chondrocyte adhesion to the microsphere surface. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into athymic mouse subcutaneous sites. Chondrocyte transplantation without PLGA microspheres and PLGA microsphere implantation without chondrocytes served as controls. Four and 9 weeks after implantation, chondrocytes implanted with PLGA microspheres formed solid, white cartilaginous tissues, whereas no gross evidence of cartilage tissue formation was noted in the control groups. Histological analysis of the implants by hematoxylin and eosin staining showed mature and well-formed cartilage. Alcian blue/safranin O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosaminoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for in vivo cartilage tissue engineering. This scaffold may be useful to regenerate cartilaginous tissues through minimally invasive surgical procedures in orthopedic, maxillofacial, and urologic applications.     

Biodegradable polymeric scaffolds for musculoskeletal tissue engineering

Biodegradable scaffolds have played an important role in a number of tissue engineering attempts over the past decade. The goal of this review article is to provide a brief overview of some of the important issues related to scaffolds fabricated from synthetic biodegradable polymers. Various types of such materials are available; some are commercialized and others are still in the laboratories. The properties of the most common of these polymers are discussed here. A variety of fabrication techniques were developed to fashion polymeric materials into porous scaffolds, and a selection of these is presented. The very important issue of scaffold architecture, including the topic of porosity and permeability, is discussed. Other areas such as cell growth on scaffolds, surface modification, scaffold mechanics, and the release of growths factors are also reviewed. A summary outlining the common themes in scaffold-related science that are found in the literature is presented.     

Scaffolds for tissue engineering of cartilage

Articular cartilage lesions resulting from trauma or degenerative diseases are commonly encountered clinical problems. It is well-established that adult articular cartilage has limited regenerative capacity, and, although numerous treatment protocols are currently employed clinically, few approaches exist that are capable of consistently restoring long-term function to damaged articular cartilage. Tissue engineering strategies that focus on the use of three-dimensional scaffolds for repairing articular cartilage lesions offer many advantages over current treatment strategies. Appropriate design of biodegradable scaffold conduits (either preformed or injectable) allow for the delivery of reparative cells bioactive factors, or gene factors to the defect site in an organized manner. This review seeks to highlight pertinent design considerations and limitations related to the development, material selection, and processing of scaffolds for articular cartilage tissue engineering, evidenced over the last decade. In particular, considerations for novel repair strategies that use scaffolds in combination with controlled release of bioactive factors or gene therapy are discussed, as are scaffold criteria related to mechanical stimulation of cell-seeded constructs. Furthermore, the subsequent impact of current and future aspects of these multidisciplinary scaffold-based approaches related to in vitro and in vivo cartilage tissue engineering are reported herein.     

微载体培养技术在骨与软骨组织工程研究中的应用☆

背景：骨与软骨组织工程学中增殖种子细胞和保持细胞特定表型是其难点,微载体生物反应器培养系统提供了很好的条件来解决这个问题。 目的：分析近年来国内外骨、软骨细胞微载体培养的研究进展,为骨与软骨细胞微载体培养技术和组织工程研究提供理论基础。 方法：由第一作者在2010-11进行检索。检索数据库：PubMed数据库(网址http://www.ncbi.nlm.gov/PubMed);万方数据库(网址http://www.wanfangdata.com.cn),资料的检索时间范围为1967/2011。英文检索词为“microcarrier,cartilage,tissue engineering”,中文检索词为“微载体,软骨,组织工程学”。排除与本文无关及陈旧、重复的文章,共保存32篇文献做进一步分析。 结果与结论：在微载体培养系统中,可较好的调控骨与软骨细胞培养条件,能在短时间内大量的增殖,并能保持其细胞的表型,甚至出现表型增强现象,在骨、软骨组织工程学研究和临床应用中有着巨大潜力。     

Novel fabrication techniques to produce microspheres by thermally induced phase separation for tissue engineering and drug delivery

A novel application of thermally induced phase separation (TIPS) is described enabling the rapid formation of monodisperse porous microspheres. By taking advantage of TIPS processing parameters, the porosity, the pore morphology (bimodal/channel-like/radial towards the centre) and the presence of an open-pore or dense skin region can be tailored. Achievable sizes range from 10 to 2000microm in diameter. The technique facilitates the homogeneous inclusion of particulate fillers and drugs. Moreover, the combined TIPS/oil-in-water emulsion technique allows for the production of microspheres with isotropic pore morphology with interconnected spherical pores of 30-70microm and well-formed porous microspheres of 10-200microm in diameter with an open porous surface. This method is advantageous over existing techniques by avoiding the use of long-term exposure to an aqueous continuous phase as used in oil-in-water or water-in-oil-in-water processing and therefore drug encapsulation efficiencies will be higher.     

Biodegradable honeycomb collagen scaffold for dermal tissue engineering

Tissue engineering requires a mechanically stable, biocompatible, and biodegradable scaffold that permits cell adherence and proliferation, allows preservation of cell-specific properties, and suitable for surgical implantations. In this study, honeycomb collagen sheet was used for three-dimensional (3D) cultures of human skin fibroblasts and characterized as an effective and suitable scaffold for dermal tissue engineering. About 1-mm-thick honeycomb collagen sheets, prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation, and sterilized by heat, were placed on the proliferating fibroblasts on day 3 of the culture. The cells attached quickly to the collagen scaffold, proliferated inside the honeycomb pores, and formed a structure similar to dermis within 60 days. On day 60, total cellular DNA content of the 3D cultures was 12-fold higher when compared with the 2D control cultures without the scaffold. Measurement of procollagen type I in the media demonstrated a 20-fold increase. Scanning electron microscopy of the 3D cultures showed a well-formed structure similar to dermis and biodegradation of the honeycomb collagen scaffold. Our study proved that honeycomb collagen sheet is a mechanically stable, biocompatible and biodegradable scaffold for dermal tissue engineering, and also potentially useful for other cell-based therapies and tissue engineering applications.     

Preadipocyte seeded PLGA scaffolds for adipose tissue engineering.

Adipose tissue equivalents have not been addressed as yet despite the clinical need in congenital deformities, posttraumatic repair, cancer rehabilitation, and other soft tissue defects. Preadipocytes were successfully harvested from rat epididymal fat pads of Sprague-Dawley and Lewis rats and expanded ex vivo. In vitro cultures demonstrated full differentiation of preadipocytes into mature adipocytes with normal lipogenic activity. The onset of differentiation was well-controlled by regulating preadipocyte confluency. Poly(lactic-co-glycolic) acid (PLGA) polymer disks with 90% porosity, 2.5 mm thick, 12 mm diameter, pore size range of 135-633 microm were fabricated and seeded with preadipocytes at 10(5) cells/mL. Disks in vitro demonstrated fully differentiated mature adipocytes within the pores of the disks. Short-term in vivo experiments were conducted by implanting preseeded disks subcutaneously on the flanks of rats for 2 and 5 weeks. Histologic staining of harvested disks with osmium tetroxide (OsO4) revealed the formation of adipose tissue throughout the disks. Fluorescence labeling of preadipocytes confirmed that formed adipose tissue originated from seeded preadipocytes rather than from possible infiltrating perivascular tissue. This study demonstrates the potential of using primary preadipocytes as a cell source in cell-seeded polymer scaffolds for tissue engineering applications.     

Review: tissue engineering for regeneration of articular cartilage

Joint pain due to cartilage degeneration is a serious problem, affecting people of all ages. Although many techniques, often surgical, are currently employed to treat this affliction, none have had complete success. Recent advances in biology and materials science have pushed tissue engineering to the forefront of new cartilage repair techniques. This review seeks to condense information for the biomaterialist interested in developing materials for this application. Articular cartilage anatomy, types of injury, and current repair methods are explained. The need for biomaterials, current commonly used materials for tissue-engineered cartilage, and considerations in scale-up of cell-biomaterial constructs are summarized.     

Introduction to tissue engineering and application for cartilage engineering

Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and molecular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general components are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreograph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs or tissues. Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development, grafting and safety studies.     

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.     

Cellulose-based scaffold materials for cartilage tissue engineering

Non-woven cellulose II fabrics were used as scaffolds for in vitro cartilage tissue engineering. The scaffolds were activated in a saturated Ca(OH)(2) solution and subsequently coated with a calcium phosphate layer precipitated from a supersaturated physiological solution. Chondrocyte cell response and cartilage development were investigated. The cell adherence was significantly improved compared to untreated cellulose fabrics, and the proliferation and vitality of the adhered chondrocytes were excellent, indicating the biocompatibility of these materials. A homogeneous distribution of the seeded cells was possible and the development of cartilageous tissue could be proved. In contact with a physiological chondrocyte solution, calcium is expected to be leached out from the precipitated layer, which might lead to a microenvironment that triggers the development of cartilage in a way similar to cartilage repair in the vicinity of subchondral bone.     

RGD-peptides for tissue engineering of articular cartilage

One keypoint in the development of a biohybrid implant for articular cartilage defects is the specific binding of cartilage cells to a supporting structure. Mimicking the physiological adhesion process of chondrocytes to the extracellular matrix is expected to improve cell adhesion of in vitro cultured chondrocytes. Our approach involves coating of synthetic scaffolds with tailor-made, cyclic RGD-peptides, which bind to specific integrin receptors on the cell surface. In this study we investigated the expression pattern of integrins on the cell surface of chondrocytes and their capability to specifically bind to RGD-peptide coated materials in the course of monolayer cultivation. Human chondrocytes expressed integrins during a cultivation period of 20 weeks. Receptors proved to be functionally active as human and pig chondrocytes attached to RGD-coated surfaces. A competition assay with soluble RGD-peptide revealed binding specificity to the RGD-entity. Chondrocyte morphology changed with increasing amounts of cyclic RGD-peptides on the surface.     

软骨组织工程生物反应器的研究进展

体外软骨构建是软骨组织工程产业化发展及临床应用的重要手段。然而,采用现有的体外构建技术无法构建功能接近正常的软骨。生物反应器能够在一定程度上模拟体内环境,有望弥补现有体外构建技术的弊端。研究发现,流体剪切力、静态液压力和直接压缩力是体内软骨发育和成熟的重要力学因素,常用软骨生物反应器均据此设计而产生。由于不同类型生物反应器各具特点,研究和开发新型复合式生物反应器将成为未来的发展方向。对目前软骨组织工程生物反应器的研究现状做一综述。     

细胞外基质来源支架在软骨组织工程中的应用

BACKGROUND: At present, a variety of extracellular matrix-derived scaffolds have been successfully applied for cartilage tissue engineering in experiment and clinical practice. OBJECTIVE: To summarize the application and research status of extracellular matrix-derived scaffolds in cartilage tissue engineering. METHODS: A computer-based online search in PubMed, CNKI, CqVip and WanFang databases was performed using the keywords of “tissue engineering, cartilage, extracellular matrix, scaffolds” in English and Chinese, respectively. A total of 1 140 literatures were retrieved, and finally 65 eligible literatures were included. RESULTS AND CONCLUSION: In terms of the components, extracellular matrix-derived scaffolds are divided into monomeric natural polymers, mixed natural polymers, natural polymers compositing with synthetic polymers as well as acellular extracellular matrix-derived materials. Extracellular matrix-derived scaffolds hold good biocompatibility and degradability, and can promote proliferation and differentiation of choncrodytes; therefore, they as good bionic scaffolds have been applied for cartilage tissue engineering in clinical practice, However, poor mechanical properties and difficulty to molding should never be ignored. Further research should focus on improving the preparation technology by combining synthetic materials with extracellular matrix-derived scaffolds for cartilage tissue engineering.