In vitro growth and activity of primary chondrocytes on a resorbable polylactide three-dimensional scaffold

Sheep articular chondrocytes were cultured for 3, 6, and 9 weeks on a three-dimensional porous scaffold from poly(L/DL-lactide) 80/20%. Cell growth and activity was estimated from the amount of proteoglycans attached to the polylactide scaffold and the amounts of DNA and proteins measured in the cell lysate. Cell morphology was assessed from scanning electron microscopy. Histochemical staining of proteoglycans present in the sponge was used to visualize the chondrocyte ingrowth in the scaffold. The amounts of DNA, proteins, and proteoglycans increased with time of culturing. Chondrocytes on the polylactide scaffold maintained their round shape. The cell ingrowth into the sponge progressed with time of culturing and proceeded from the upper surface of the sponge toward its lower surface. At 9 weeks, the chondrocytes filled the whole scaffold and reached the opposite side of the sponge. The proteoglycans network was, however, more dense at the upper half of the scaffold.     

Enhanced expression of hyaluronic acid in osteoarthritis-affected knee-cartilage chondrocytes during three-dimensional in vitro culture in a hyaluronic-acid-retaining polymer scaffold

BackgroundChondrocyte transplantation to address cartilage damage is an established solution. Because hyaluronic acid (HA) is an essential component for homeostasis of the cartilage, in order to arrive at methodologies to utilize its advantages in cell-based therapies, we compared the HA retention capability of a thermoreversible gelation polymer scaffold-based environment (3D-TGP) with conventional in vitro cell culture methodologies.MethodsChondrocytes derived from osteoarthritis-affected knee joint cartilage of elderly patients were used and accomplished in three phases. In Phase I, the levels of HA secreted by chondrocytes were measured in culture supernatant. In Phase II, retention capacity of externally added HA was quantified indirectly by measuring the HA released in culture supernatant, and in Phase III, the expression of CD44 on cells was analysed by immunohistochemistry.ResultsIn Phase I, the average HA in the 3D supernatant was 3% that of 2D. In phase II, 80% of externally added HA was detected in the 2D on day 7, while in 3D-TGP, only 0.1% was released until day 21. In Phase III, 2D yielded individual cells that started degenerating from the third week; in 3D-TGP cells grew for a longer duration, formed a tissue-like architecture with extracellular matrix with significantly intense staining of CD44 than 2D.ConclusionThe capability of the 3D-TGP culture environment to retain HA and support chondrocytes to grow with a tissue-like architecture expressing higher HA content is considered advantageous as it serves as an in vitro culture platform that enables tissue engineering of cartilage tissue with native hyaline phenotype and higher HA expression. The in vitro environment being conducive, based on this data, we also recommend that the TGP be tried as an encapsulation material in clinical studies of chondrocyte implantation for optimal clinical outcome.     

In vitro culture of rabbit growth plate chondrocytes. 2. Chondrodystrophic mutants

The epiphyseal plates of newborn normal (Cd/?), dwarf (Dw/Dw) and achondroplastic (ac/ac) rabbits were grown in organ culture for 48 hours. The 3H-thymidine labeling index of proliferating zone chondrocytes from both mutants was below that of normals. The generation time of Dw/Dw animals was longer than that of normals (greater than 24 vs. 17 hours respectively). Inasmuch as the medium was supplemented with 10% fetal calf serum, which presumably contains somatomedin, these data suggest that the Dw/Dw mutant cannot be considered simply a model for pituitary dwarfism. Pituitary fibroblast growth factor (FGF), 50ng/ml, decreased the labeling index of chondrocytes from both Dw/Dw and ac/ac animals. Thus neither a lack of FGF nor an end-organ non-responsiveness to this factor seems to play a role in the pathogenesis of either growth disturbance.     

含镁多孔支架材料的体外抗菌活性和生物相容性

文题释义：快速成型：是一种材料加工方法,它是在现代CAD/CAM技术、激光技术、计算机数控技术、精密伺服驱动技术及新材料技术的基础上集成发展起来的。不同种类的快速成型系统因所用成形材料不同,成形原理和系统特点也各有不同,但基本原理都是“分层制造,逐层叠加”。 聚乳酸：是以乳酸为主要原料聚合得到的聚合物,具有良好的生物降解性、生物相容性以及延展性,但其机械强度不足,且降解后会产生酸性代谢产物,限制了其应用范围,常与其他一种或多种生物材料复合使用,以增强骨生物活性或生物力学强度。 背景：将多聚物材料与生物陶瓷材料复合制成有机/无机复合三维支架材料,可赋予支架骨传导所必需的理化特性,同时强化材料的力学性能,但大多数骨替代材料无法预防缺损部位的感染。研究发现由于镁的降解可产生局部碱性环境,使镁具有一定的抗菌活性。 目的：探讨含镁多孔支架材料的体外抗菌活性和细胞相容性。 方法：应用低温快速成型技术制备聚乳酸/β-磷酸三钙/镁多孔支架材料,其中β-磷酸三钙与镁的质量比分别为2∶1和1∶2,分别设为PTM(2∶1)组、PTM(1∶2)组;同时应用低温快速成型技术制备聚乳酸与聚乳酸/β-磷酸三钙多孔支架材料,分别设为P组、PT组。检测4组支架的表面形貌、孔径、孔隙率及压缩模量。将金黄色葡萄球菌(ATCC 35923)接种于4组支架表面24 h,通过涂板计数法和激光共聚焦显微镜观察材料的抗菌活性。将小鼠前成骨细胞MC3T3-E1分别与4组支架材料共培养,通过CCK-8法分析材料对细胞黏附和增殖的影响。 结果与结论：①4组支架材料表面都形成相对均匀的多孔结构,4组支架间孔径大小和孔隙率比较差异均无显著性意义(P > 0.05);②PTM(2∶1)组和PTM(1∶2)组压缩模量明显高于P组、PT组(P < 0.05),PTM(1∶2)组明显高于PTM(2∶1)组(P < 0.05);③涂板计数实验显示,PTM(2∶1)组、PTM(1∶2)组菌落形成单位明显低于P组、PT组(P < 0.05),其余组间比较差异无显著性意义(P > 0.05);④培养6 h,PT组、PTM(2∶1)组、PTM(1∶2)组黏附细胞数量多于P组(P < 0.05),PTM(2∶1)组和PTM(1∶2)组比较差异均无显著性意义(P > 0.05);⑤培养1 d时,仅PT组细胞增殖优于P组(P < 0.05);培养4,7 d时,PT组、PTM(2∶1)组、PTM(1∶2)组细胞增殖均优于P组(P < 0.05),PTM(2∶1)组和PTM(1∶2)组比较差异均无显著性意义(P > 0.05);⑥结果表明,聚乳酸/β-磷酸三钙/镁多孔支架材料不但具有良好的抗菌活性,而且具有优良的细胞相容性和一定的抗压能力。 ORCID: 0000-0002-3367-674X(马瑞) 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     

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.     

In vitro tendon cell growth rates on a synthetic fiber scaffold material and on standard culture plates

Growth rates of rat tendon fibroblasts cultured in a three-dimensional carbon fiber matrix were compared with those of cells cultured on standard flat culture plates. The carbon fiber has been used as a tissue scaffold for tendon and ligament repair in animal and clinical studies. While cell growth on the culture plates appears to follow a growth curve containing a lag phase, a log phase, and plateau phase of growth, cell growth in the fiber matrix was characterized by a suppressed log phase of growth. SEM and cytotoxicity studies indicated that this effect was not caused by growth-inhibiting or cytotoxic substances from the carbon fiber. While we cannot rule out the possibility that cell growth was influenced by the surface chemistry of the carbon substrate, evidence from this and other studies suggests that the observed effect was caused by a lack of readily available surface area for cell attachment and growth on the small fibers. Because cell colonies growing on individual fibers are limited (at least in theory) to growing in two directions only, they enjoy limited opportunities for cell migration and growth--in contrast with cell colonies on flat culture plates. These results suggest fundamental differences in the mechanisms controlling cell growth on planar vs. three-dimensional fiber substrates.     

The growth of chondrocytes into a fibronectin-coated biodegradable scaffold

Porous scaffolds made from a biodegradable copolymer of trimethylene carbonate and glycolide were evaluated for tissue-engineered medical products. We examined the scaffold coated with cell adhesion protein and fibronectin and cultured under a dynamic mixing condition to enhance the growth of chondrocytes. Our hypothesis was that the combination of coating and dynamic mixing would be beneficial to the viability of the chondrocytic cells. Fibronectin was selected as the model protein because of its availability and routine assaying methods. Sterile samples of scaffolds of about 1 mm in thickness were coated with fibronectin at 37 degrees C for 1.5 h. Four groups of scaffolds were used: uncoated static or dynamic, and coated static or dynamic. Scaffold samples were placed in either a Petri dish or a spinner flask (static vs. dynamic groups) after inoculation with rat chondrocytes of an initial cell density of 1.29 x 10(5) cell/mL. After 7, 14, 21, and 28 days, each sample was fixed, embedded, and sectioned at 5 micro thickness. The sections were double-label immunostained using antibodies against cellular fibronectin synthesized by adherent cells as a measure of cell viability. A Hoechst 33258 nuclear stain was used to measure the number of cells attached to the scaffold at each time interval. The slides were examined using a fluorescence microscope to determine the cell ingrowth. At least 25 fields/treatment group (except the 7 day group) were measured. The data showed that cell in-growths into the porous scaffolds were higher at all time periods for the coated dynamic group than those for the other three groups.     

In vitro cellular responses to scaffolds containing two microencapulated growth factors

Growth factors play an important role in the complex cascade of tissue events in periodontal regeneration, although optimal methods of delivery remain to be identified. We hypothesize that multiple delivery of growth factors, particularly via a microparticle-containing scaffold, will enhance cellular events leading to periodontal regeneration. In this study, cellular responses of periodontal ligament fibroblasts (PDLFs) in scaffolds containing microparticles (MPs) loaded with either bone morphogenetic protein (BMP)-2, insulin-like growth factor (IGF)-1, or a mixture of both MPs were evaluated, and the dual-MP-containing scaffold exhibited the release of different proteins in a sustained and independent fashion. When PDLF-seeded scaffolds were cultured in a flow perfusion bioreactor, cell metabolism and proliferation of PDLFs were significantly increased within 3 days in all IGF-1-containing scaffolds compared with those in groups lacking IGF-1 and particulate delivery enhanced these effects between 3 and 7 days. The dual-MP-containing group showed the most positive results. Both the BMP-2-in-MP and IGF-1-in-MP groups showed greater effects of alkaline phosphatase activity, more osteocalcin and osteopontin production, and more calcium deposition compared with matched GF-adsorbed groups. All osteoblastic markers were at their highest in the dual-MP-containing group at all detected time points. The combined results suggest that our dual-MP-containing scaffold can be used as a cell vehicle to positively affect cell behavior, thus exhibiting the potential to be a candidate scaffold for future periodontal tissue engineering.     

The effect of polymer composition on the gelation behavior of PLGA-g-PEG biodegradable thermoreversible gels

Graft copolymers consisting of a poly(D,L-lactic acid-co-glycolic acid) backbone grafted with polyethylene glycol side chains were synthesized and formed thermoreversible gels in aqueous solutions that exhibited solution behavior at low temperature and sol-to-gel transitions at higher temperature. The composition of the polymer and relative amounts of polylactic acid, glycolic acid, and ethylene glycol were varied by controlling the precursor concentrations and reaction temperature. The gelation temperature could be systematically tailored from 15 to 34 degrees C by increasing the concentration of polyethylene glycol in the graft copolymer. The gelation temperature also depended on the polymer molecular weight and concentration. This work has importance for the development of water soluble gels with tailored compositions and gelation temperatures for use in tissue engineering and as injectable depots for drug delivery.     

Porous chitosan-gelatin scaffold containing plasmid DNA encoding transforming growth factor-beta1 for chondrocytes proliferation

Cartilage defects as a result of disease or injury have a very limited ability to heal spontaneously. Recently, tissue engineering and local therapeutic gene delivery systems have been paid much attention in the cartilage natural healing process. Gene-activated matrix (GAM) blends these two strategies, serving as local bioreactor with therapeutic agents expression and also providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In the current study, we used chitosan-gelatin complex as biomaterials to fabricate three-dimensional scaffolds and plasmid DNA were entrapped in the scaffolds encoding transforming growth factor-beta1 (TGF-beta1), which has been proposed as a promoter of cartilage regeneration for its effect on the synthesis of matrix molecules and cell proliferation. The plasmid DNA incorporated in the scaffolds showed a burst release in the first week and a sustained release for the other 2 weeks. The gene transfectd into chondrocytes expresses TGF-beta1 protein stably in 3 weeks. The histological and immunohistochemical results confirmed that the primary chondrocytes cultured into the chitosan-gelatin scaffold maintained round and owned characters of high secretion of specific ECM. From this study, it can be concluded that this gene-activated chitosan-gelatins matrix has a potential in the application of cartilage defects regeneration.     

In vitro studies on clonal growth of chondrocytes in thanatophoric dysplasia

Thanatophoric dysplasia (TD) is characterized by a disorganized growth plate with markedly reduced proliferative and hypertrophic cartilage zones. Therefore, we studied in vitro the proliferation rates of articular chondrocytes from five TD patients and age-matched controls in response to bFGF, IGF-I, IGF-II, and TGF-β1. In human fetal controls bFGF was the most potent growth factor. Clonal growth of articular chondrocytes in response to bFGF was reduced in two of five TD patients and slightly below the range of controls in a third case. Stimulation of chondrocyte proliferation by IGF I and II was reduced in the patient whose response to bFGF was most markedly impaired. The effect of TGF-β1 ranged from normal to slightly elevated values in TD fetuses. These results indicate heterogeneity of the underlying defects in TD. Low proliferative responses of chondrocytes to bFGF and IGF-I/II are likely to play a key role in the pathogenesis of some cases. In two of five patients studied, the mechanisms of bFGF and IGF-signal transduction are candidates for the primary molecular defect. © 1996 Wiley-Liss, Inc.     

BMP-2 embedded atelocollagen scaffold for tissue-engineered cartilage cultured in the medium containing insulin and triiodothyronine--a new protocol for three-dimensional in vitro culture of human chondrocytes

When the chondrocytes are isolated from the native cartilage and proliferate in vitro, they soon lose their original ability to express glycosaminoglycan (GAG) and type II collagen, which is termed dedifferentiation, or decrease cell viability. We first examined in vitro cartilage regeneration of tissue-engineered pellets that consisted of human auricular chondrocytes and atelocollagen and that were incubated in vitro under stimulation with bone morphogenetic protein-2 (BMP-2), insulin, and T(3). We then examined the administration of those growth factors into the scaffold or in the medium and explored the possibility that the atelocollagen, the hydrogel scaffold of the chondrocytes, may function for drug delivery of the factors. BMP-2 in the atelocollagen with the supplement of insulin and T3 in the medium could not only produce a greater GAG matrix in a shorter period but also sustain cell viability with lower mortality. The insulin in the medium could be better administered only for 2 weeks, rather than 3 weeks, which would save time and cost, hence shortening the in vitro culture of chondrocytes. Our protocol of mixing BMP-2 into the atelocollagen with the supplement of insulin and T3 hormone might provide a new insight into the development of tissue engineering in chondrogenesis.     

The use of growth factors in the proliferation of avian articular chondrocytes in a serum-free culture system

The purpose of this research was to develop a serum-free culture system for the proliferation of articular chondrocytes. Various growth factors and hormones were tested for their ability to stimulate avian articular chondrocyte proliferation in a defined, serum-free media. Multiple members of the fibroblast growth factor (FGF) family (FGFs: 2, 4, and 9), insulin-like growth factor-1 (IGF-1) and transforming growth factor beta (TGF-beta) significantly stimulated H-thymidine uptake by chondrocytes grown in an adherent serum-free, culture system. Double or triple combinations of these mitogenic growth factors further stimulated cell proliferation to levels that were equivalent to, or surpassed those of cells grown in serum. Although proliferation was maximally stimulated, chondrocytes grown in the presence of FGF-2, IGF-1, and TGF-beta, began to exhibit changes in morphology and collagen II expression declined. This culture system could be used to rapidly expand a population of articular chondrocytes prior to transferring these cells to a non-adherent culture system, which could then stabilize the chondrocyte phenotype and maximize matrix synthesis and integrity.     

去细胞化全肝生物支架循环灌注培养条件下体外细胞再植*

背景：通过去细胞化技术制备全肝生物支架成为缓解供体短缺的新技术,优化肝脏组织的去细胞化流程成为新的课题。 目的：通过去细胞化技术建立完整保留肝脏三维结构和脉管系统的全肝生物支架,利用自主设计的循环灌注培养装置实现生物支架体外细胞再植。 方法：通过门静脉路径循环灌注去垢剂Triton X-100,十二烷基硫酸钠,并用磷酸盐缓冲液洗脱残留去垢剂。通过动态循环灌注培养装置进行支架与HepG2细胞的共培养,观察植入细胞在全肝生物支架内的功能表达。 结果与结论：经去垢剂灌注后,支架呈现保持肝脏三维结构的透明结构,苏木精-伊红染色以及扫描电镜结果显示细胞成分被完全移除,Masson’s Trichrome染色可见大量胶原纤维,免疫组化结果证实纤维连接蛋白和层粘连蛋白保存完整。循环灌注培养下细胞白蛋白表达量及尿素合成量较平板培养明显提高。说明去细胞化肝脏生物支架可作为体外肝脏组织重建的基础材料,动态循环灌注方法可实现支架中细胞再植。      

In vitro culture of rabbit growth plate chondrocytes. 1. Age-dependence of response to fibroblast growth factor and "chondrocyte growth factor"

The growth-promoting effects of pituitary fibroblast growth factor (FGF) and "chondrocyte growth factor" (CGF), a contaminant of NIH-bovine TSH and ovine LH, were studied in monolayer and/or organ culture of epiphyseal plate chondrocytes of rabbits 1 day to 10 weeks old. The response to FGF (50 ng/ml) and CGF(TSH) and CGF(LH) (64 micrograms/ml) was age-dependent. In cultures from animals aged 4 weeks or older, the growth factors consistently stimulated DNA synthesis while decreasing incorporation of radiosulfate into matrix macromolecules. In organ cultures of the growth plate of rabbits less than 1 week old, FGF and NIH-TSH and LH actually diminished rather than promoted incorporation of 3H-thymidine. In organ culture the generation times of newborn rabbit proliferating zone chondrocytes, measured in vivo and in vitro by nuclear grain count dilution in 3H-thymidine autoradiographys, were 11 and 16 to 17 hours respectively. FGF and LH increased the generation time of 1- and 4-day old rabbit chondrocytes from 16 to over 24 hours. The stimulatory effect of CGF(TSH) in the older age group was much greater than that of NIH(LH). The dog-dose response curves of FGF and CGF(LH) were parallel, supporting Jones and Addison's view that the CGF activity of CGF(LH) derives from its content of FGF.     

犬关节软骨细胞的体外分离与培养

背景：自1967年Manning等提出的通过胰蛋白酶和细菌胶原酶联合消化法分离软骨细胞以来,软骨细胞的体外分离培养研究较多,但目前尚无统一标准。 目的：研究犬关节软骨细胞在体外分离以及培养的条件,寻求体外扩增软骨细胞较简便、可行、高效的实验方法。 方法：取3周龄幼犬关节软骨组织,应用胰酶、胶原酶制定8种消化方法获取软骨细胞,对比不同方法所获取细胞数量及细胞成活率,分离细胞进行原代及传代培养,观察各代软骨细胞形态。 结果与结论：在体外分离犬关节软骨细胞的各种方法中,单纯应用Ⅱ型胶原酶消化法获得的软骨细胞数最多,细胞成活率最高。软骨细胞可以通过体外培养获得扩增,并且维持良好的细胞形态及表型,但仅限于5代以内。     

胶原复合梯度磷酸三钙负载软骨细胞的体外培养

目的 观察新型三维支架材料胶原复合梯度磷酸三钙在体外与软骨细胞的相容性和黏附性,评价其作为软骨组织工程支架的可行性.方法 取8周龄新两兰大白兔膝关节软骨,以酶消化法获得高纯度软骨细胞,培养3代后与三维支架材料胶原复合梯度磷酸三钙在体外复合培养.用倒置相差显微镜、HE染色、免疫组织化学及扫描电镜观察软骨细胞形态、Ⅱ型胶原表达及成软骨能力,同时观察支架材料与软骨细胞的相容性.结果 扫描电镜观察显示支架材料具有疏松多孔结构,孔隙结构规则,孔径100～150 μm,材料内部孔与孔之间贯通良好.支架亲水性好.软骨细胞吸附于支架表面,增殖并逐渐顺孔隙迁徙至支架内部,在孔壁贴附良好,表型维持稳定,可分泌细胞外基质.结论 胶原复合梯度磷酸三钙三维支架具有良好的细胞相容性.