透明质酸改性壳聚糖-胶原-羟基磷灰石复合支架力学特性及成骨细胞的增殖

背景：组织工程中,种子细胞需依赖于细胞外基质的存在才能发挥功能。因此支架材料的选择具有重大意义。 目的：制备一种新型改性壳聚糖-胶原-羟基磷灰石复合支架,优化易于细胞黏附的组织工程支架材料工艺。 方法：壳聚糖与透明质酸进行交联,红外和差示扫描量热图谱检测其结构;改性壳聚糖与胶原按1∶2,1∶1和2∶1制备3种改性壳聚糖-胶原-羟基磷灰石复合支架,将复合支架与成骨细胞MC3T3-E1联合培养,CCK-8法检测增殖,绘制生长曲线。 结果与结论：透明质酸和壳聚糖以酰胺键形成交联的新化合物,孔径在50~250 μm之间,孔隙率随着胶原水平、弹性模量的增加而增加,而密度则减少;增加胶原的含量在细胞联合培养初期有利于细胞对支架的黏附和增殖,但从第10天开始,3种样品中细胞数量相差不大,均出现平台期;苏木精-伊红染色发现成骨细胞在培养初期沿着支架材料内部空隙贴壁生长,随着培养天数的增加,贴壁细胞呈集落样生长,可明显看到细胞间连接。说明透明质酸改性壳聚糖/胶原/纳米羟基磷灰石复合材料可以作为骨支架材料供成骨细胞黏附、增殖,其中胶原与壳聚糖的体积比为1∶1为较优配比。     

三种高分子支架对骨髓基质细胞生长影响的研究

研究高分子复合支架聚乳酸均聚物(PLA)、聚乳酸/聚乙二醇共聚物(PLA-PEG)和聚乳酸/聚乙醇酸共聚物(PLGA)对骨髓基质细胞(M SC)黏附、增殖和分化的影响。分离、纯化兔骨髓基质细胞,分别和3种支架材料共培养。在不同时间段,应用细胞计数,碱性磷酸酶(ALP)定量分析,四环素荧光染色,扫描电镜,RT-PCR的方法,观察细胞在3种材料上生长,黏附和矿化沉积情况。结果表明细胞在3种支架材料上生长良好,PLGA上细胞生长的数量最多,其次是PLA-PEG、PLA,呈时间依赖性增加。3种材料上的细胞均表达ALP活性,PLA-PEG和PLGA组的ALP活性无差异,但显著高于PLA组,差异有显著性。RT-PCR反应可检测到骨钙素和纤维I型胶原在mRNA水平的表达。扫描电镜见细胞最初呈球形附着于材料上,7 d后细胞数量增加并分泌细胞外基质。两周后可见钙化结节形成,四环素荧光呈黄绿色染色。结果提示M SC细胞能较好的黏附于3种聚合物支架上,生长良好,并表达一定的成骨潜能,但PLA共聚物PLA-PEG和PLGA优于PLA均聚物,有望成为较好的构建组织工程骨的支架。     

Preparation,characterization and in vitro analysis of novel structured nanofibrous scaffolds for bone tissue engineering

In a previous study, a three-dimensional nanofibrous spiral scaffold for bone tissue engineering was developed, which showed enhanced human osteoblast cell attachment, proliferation and differentiation compared with traditional cylinder scaffolds, owing to the incorporation of spiral structures and nanofiber. However, the application of these scaffolds to bone tissue engineering was limited by their weak mechanical strength. This limitation triggered the design for novel structured scaffolds with reinforced physical characteristics. In this study, spiral polycaprolactone (PCL) nanofibrous scaffolds were inserted into poly(lactide-co-glycolide) (PLGA) microsphere sintered tubular scaffolds to form integrated scaffolds to provide mechanical properties and bioactivity appropriate for bone tissue engineering. Four experiment groups were designed: PLGA cylinder scaffold; PLGA tubular scaffold; PLGA tubular scaffold with PCL spiral structured inner core; PLGA tubular scaffold with PCL nanofiber containing spiral structured inner core. The morphology, porosity and mechanical properties of the scaffolds were characterized. Furthermore, human osteoblastic cells were seeded on these scaffolds, and the cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds were evaluated. The integrated scaffolds had Young’s modulus 250–300 MPa, and compressive strength 8–11 MPa under uniaxial compression. With the addition of an inner highly porous insert to the tubular shell, human osteoblast cells seeded on the integrated scaffolds showed slightly higher cell proliferation, 20–25% more alkaline phosphatase expression and twofold higher calcium deposition than those on the cylinder and tubular scaffolds. Furthermore, compared with sintered PLGA cylinder scaffolds, the integrated scaffolds allowed better cellular infiltration Therefore, this design demonstrates great potential for integrated scaffolds in bone tissue engineering applications.     

Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering

Hyaluronic acid (hyaluronan, HA) was immobilized onto the surface of macroporous biodegradable poly(D,L-lactic acid-co-glycolic acid) [PLGA] scaffolds to enhance the attachment, proliferation, and differentiation of chondrocytes for cartilage tissue engineering. The PLGA scaffolds were prepared by blending PLGA with varying amounts of amine-terminated PLGA-PEG di-block copolymer. They were fabricated by a gas foaming/salt leaching method. HA was chemically conjugated to the surface-exposed amine groups on the pre-fabricated scaffolds. The amount of surface exposed free amine groups was quantitatively determined by conjugating an amine-reactive fluorescent dye to the PLGA blend films. The extent of HA immobilization was also confirmed by measuring water contact angles. When chondrocytes were seeded within HA modified PLGA scaffolds, enhanced cellular attachment was observed compared to unmodified PLGA scaffolds. Furthermore, glycosaminoglycan and total collagen synthesis increased substantially for HA modified PLGA scaffolds. RT-PCR result and histological examination of the resultant cartilage tissue revealed that HA modified scaffolds excelled in inducing cartilage tissue formation in terms of collagen type II expression and tissue morphological characteristics.     

Biological behavior of pre-osteoblasts on natural hydroxyapatite: a study of signaling molecules from attachment to differentiation

Several biomaterials have been widely used in bone regeneration in both orthopedic and oral surgeries. However, it is poorly understood how these biomaterials alter osteoblast phenotype. It prompted us to examine the involvement of signaling proteins during preosteoblast adhesion (attachment), proliferation, and differentiation on natural hydroxyapatite (HA) from bovine bone. Our results indicated that natural HA is able to promote osteoblast adhesion, proliferation, and differentiation. The osteoblast/HA interaction requires phosphorylation of tyrosine residues of focal adhesion kinase, Src, and Paxillin upon integrin activation, which culminates in the control of cofilin phosphorylation (at serine 03) via rac-1 activation. In part, these signaling pathways are responsible for actin-rearrangement, responsible to adapt cell-shape on HA particles. In regarding to osteoblast differentiation, we showed that natural HA favored extracellular matrix remodeling by stimulating matrix metalloproteinase activities and alkaline phosphatase activity. Overall, this study demonstrates that osteoblast response toward bovine bone HA is initially mediated by activation of focal adhesion components, culminating on actin-rearrangement executed by cofilin activation via rac-1. Moreover, bovine bone HA provided an excellent microenvironment for osteoblast activity, since adhesion to differentiation.     

Effect of demineralized bone particle/poly(lactic-co-glycolic acid) scaffolds on the attachment and proliferation of mesenchymal stem cells

The aim of this study was to investigate the effect of demineralized bone particle/ poly(lactic-co-glycolic acid) (DBP/PLGA) scaffolds on the proliferation of mesenchymal stem cells (MSCs). DBP/PLGA hybrid scaffolds were fabricated by solvent casting/salt-leaching with DBP contents of 0, 20, 40, and 80 wt%. MSCs were seeded on the DBP/PLGA scaffolds and then evaluated by a series of analytical process: SEM, MTT, RT-PCR, and in vivo histological assay. As the DBP contents increased, the cell attachment behavior and cell viability also increased. A DBP content of 80 wt% marked the best water absorption performance and the highest cell viability. Gene expression of aggrecan on DBP/PLGA scaffolds tended to increase, whereas that on PLGA scaffolds was decreased at 1 week. However, strong expression of aggrecan was observed at 2 weeks regardless of the contents of DBP. Scaffolds showed a trend of increasing type II and I collagen at 2 weeks. The results showed that MSCs on DBP/PLGA scaffolds showed more efficient cell proliferation and tissue formation in the presence of tissue-inductive stimuli. Suitable biomaterials could be more conducive to proliferation of MSCs. These results suggest that the DBP/PLGA scaffolds are a feasible biomaterial for intervertebral disc regeneration.     

The effect of timing of mechanical stimulation on proliferation and differentiation of goat bone marrow stem cells cultured on braided PLGA scaffolds

Bone marrow stromal cells (BMSCs) have been shown to proliferate and produce matrix when seeded onto braided poly(L-lactide/glycolide) acid (PLGA) scaffolds. Mechanical stimulation may be applied to stimulate tissue formation during ligament tissue engineering. This study describes for the first time the effect of constant load on BMSCs seeded onto a braided PLGA scaffold. The seeded scaffolds were subjected to four different loading regimes: Scaffolds were unloaded, loaded during seeding, immediately after seeding, or 2 days after seeding. During the first 5 days, changing the mechanical environment seemed to inhibit proliferation, because cells on scaffolds loaded immediately after seeding or after a 2-day delay, contained fewer cells than on unloaded scaffolds or scaffolds loaded during seeding (p<0.01 for scaffolds loaded after 2 days). During this period, differentiation increased with the period of load applied. After day 5, differences in cell content and collagen production leveled off. After day 11, cell number decreased, whereas collagen production continued to increase. Cell number and differentiation at day 23 were independent of the timing of the mechanical stimulation applied. In conclusion, static load applied to BMSCs cultured on PLGA scaffolds allows for proliferation and differentiation, with loading during seeding yielding the most rapid response. Future research should be aimed at elucidating the biomechanical and biochemical characteristics of tissue formed by BMSCs on PLGA under mechanical stimulation.     

Attachment, proliferation and differentiation of osteoblasts on random biopolyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds

Rabbit bone marrow cells were inoculated on 3D scaffolds of poly(lactic acid) (PLA), poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) to evaluate their in vitro biocompatibilities. It was found that PHBHHx had the best performance on attachment, proliferation of bone marrow cells. The cells on PHBHHx scaffolds presented typical osteoblast phenotypes: round cell shape, high alkaline phosphotase (ALP) activity, strong calcium deposition, and fibrillar collagen synthesis. After incubation for 10 days, cells grown on PHBHHx scaffolds were approximately 2x10(5)ml(-1), 40% more than that on PHB scaffolds and 60% more than that on PLA scaffolds. ALP activity of the cells grown on PHBHHx scaffolds was up to about 65U/g scaffolds, 50% higher than that of PHB and PLA, respectively. The scanning electronic microscopy (SEM) results showed that PHBHHx scaffolds had the appropriate roughness for osteoblast attachment and proliferation comparing with PHB and PLA. All these indicated that PHBHHx was a suitable biomaterial for osteoblast attachment, proliferation and differentiation from bone marrow cells.     

Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds

The effects of cell adhesion peptides (RGDS, KQAGDV, VAPG) on vascular smooth muscle cells grown on modified surfaces and in tissue-engineering scaffolds were examined. Cells were more strongly adhered to surfaces modified with adhesive ligands than to control surfaces (no ligand or a nonadhesive ligand). Cell migration was higher on surfaces with 0.2 nmol/cm(2) of adhesive ligand than on control surfaces, but it was lower on surfaces with 2.0 nmol/cm(2) of adhesive ligand than it was on control surfaces. Further, cell proliferation was lower on adhesive surfaces than it was on control surfaces, and it decreased as the ligand density increased. Similarly, in the peptide-grafted hydrogel scaffolds, cell proliferation was lower in scaffolds containing the adhesive peptides than it was in control scaffolds. After 7 days of culture, more collagen per cell was produced in control scaffolds than in scaffolds containing adhesive peptides. In addition, collagen production decreased in the scaffolds as the ligand concentration increased. While modification of a surface or scaffold material with adhesive ligands initially increases cell attachment, it may be necessary to optimize cell adhesion simultaneously with proliferation, migration, and matrix production.     

Increased osteoblast functions among nanophase titania/poly(lactide-co-glycolide) composites of the highest nanometer surface roughness

Currently, the scientific challenges for bone tissue engineering lie in the development of suitable scaffold materials that can improve bone cell adhesion, proliferation, and differentiation. The design of nanophase titania/poly(lactide-co-glycolide) (PLGA) composites offers an exciting approach to combine the advantages of a degradable polymer with nanosize ceramic particles to optimize the physical and biological properties necessary for bone regeneration. Moreover, because of the presence of nanosized ceramics, such composites can be formulated to match the surface roughness of bone. For these reasons, the objective of the present in vitro study was to investigate osteoblast (bone-forming cell) adhesion and long-term functions on nanophase titania/PLGA composites that mimic the surface roughness of bone. Various sonication powers were applied in this study to manipulate titania dispersions in PLGA and consequently control their surface roughness. Most importantly, results correlated better osteoblast adhesion and long-term functions (such as collagen, alkaline phosphatase activity, and calcium-containing mineral deposition) among nanophase titania/PLGA composites that had surface roughness values closer to natural bone. In this manner, this present study demonstrated that the nanophase titania/PLGA composites sonicated to have nanometer surface roughness values can improve osteoblast functions necessary for enhanced bone tissue engineering applications.     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

Differentiation of bone marrow stromal cells in poly(lactide-co-glycolide)/chitosan scaffolds

This study investigates the physicochemical properties of poly(lactide-co-glycolide) (PLGA)/chitosan scaffolds and the neuron growth factor (NGF)-guided differentiation of bone marrow stromal cells (BMSCs) in the scaffolds. The scaffolds were prepared by the crosslinking of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and genipin, and the differentiating BMSCs were characterized against CD44, CD90 and NeuN. The scaffold with 20% PLGA yielded 95% porosity, Young's modulus of 13 MPa, 70% adhesion of BMSCs and 1.6-fold increase in the cell viability over 7-day cultivation. BMSCs without guidance in the PLGA/chitosan scaffolds were prone to differentiate toward osteoblasts with apparent deposition of calcium. When NGF was introduced, an increased weight percentage of PLGA yielded more identified neurons. In addition, mature neurons emerged from the PLGA-rich biomaterials after induction with NGF over 2 days. A proper control over the physical and biomedical property of the scaffolds and the NGF-guided differentiation of BMSCs can be promising for nerve regeneration.     

The effect of type II collagen coating of chitosan fibrous scaffolds on mesenchymal stem cell adhesion and chondrogenesis

The biocompatibility of chitosan and its similarity to glycosaminoglycans (GAG) make it attractive for cartilage tissue engineering. We have previously reported improved chondrogenesis but limited cell adhesion on chitosan scaffolds. Our objectives were to produce chitosan scaffolds coated with different densities of type II collagen and to evaluate the effect of this coating on mesenchymal stem cell (MSC) adhesion and chondrogenesis.Chitosan fibrous scaffolds were obtained by a wet spinning method and coated with type II collagen at two different densities. A polyglycolic acid mesh served as a reference group. The scaffolds were characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and type II collagen content. Constructs were analyzed after MSCs seeding via live/dead assay, weight and DNA evaluations, SEM, and TEM. Constructs were cultured in chondrogenic medium for 21 days prior to quantitative analysis (weight, DNA, and GAG), SEM, TEM, histology, immunohistochemistry, and quantitative real time polymerase chain reaction. The cell attachment and distribution after seeding correlated with the density of type II collagen. The cell number, the matrix production, and the expression of genes specific for chondrogenesis were improved after culture in collagen coated chitosan constructs.These findings encourage the use of type II collagen for coating chitosan scaffolds to improve MSCs adhesion and chondrogenesis, and confirm the importance of biomimetic scaffolds for tissue engineering.     

Core-shell PLGA/collagen nanofibers loaded with recombinant FN/CDHs as bone tissue engineering scaffolds

Poly(lactic-co-glycolic acid) (PLGA)/collagen nanofibrous scaffolds have been utilized in the tissue engineering field. It has been shown that both fibronectin (FN) and cadherin 11 (CDH) play important roles in the progress of osteogenesis and cell adhesion. The aim of this study was to fabricate recombinant FN/CDHs (rFN/CDHs)-loaded PLGA/collagen nanofibrous scaffolds and evaluate their effects on the adhesion and differentiation of human bone marrow mesenchymal stem cells (hMSCs). PLGA/collagen nanofibers were made by coaxial electrospinning. The morphology and mechanical properties of PLGA/collagen nanofibrous mats were analyzed by scanning electron microscopy and mechanical testing, respectively. The performance of scaffolds was evaluated in terms of the viability, morphology, and osteogenic gene expression levels of hMSCs. rFN/CDHs was successfully incorporated into the PLGA/collagen nanofibers. The release of rFN/CDHs from PLGA nanofibers was investigated by liquid chromatography–mass spectrometry. rFN/CDHs improved the mechanical properties of the PLGA/collagen nanofibers. The controlled release of rFN/CDHs can enhance the proliferation of hMSCs and induce osteogenic gene expression (alkaline phosphatase, RUNX2, and osteocalcin). Our data imply that rFN/CDHs may induce hMSCs differentiation into osteoblasts and PLGA/collagen nanofibers loaded with rFN/CDHs have potential in bone tissue engineering.     

Proliferation and differentiation of human osteoblasts within 3D printed poly-lactic-co-glycolic acid scaffolds

Bone repair and regeneration can be enhanced through implantation of biocompatible and biodegradable scaffolds, which serve primarily as osteoconductive moieties. In this study, the mechanical properties and microenviroment of 3D printed poly-lactic-co-glycolic acid (PLGA) scaffolds are examined. Additionally, the proliferation and differentiation of human fetal osteoblasts are evaluated after 3 weeks of in vitro culture on the scaffolds. The results showed that the PLGA scaffolds examined had mechanical properties similar to that of trabecular bone, but was still much weaker compared to cortical bone. In addition to general porosity, the PLGA scaffolds also had micropores within macropore walls. Cultured human osteoblasts could proliferate upon seeding on the PLGA scaffolds. Alkaline phosphatase activity and osteonectin expression of the osteoblasts cultured on the PLGA scaffolds remained stable over three weeks, whilst expression of collagen type I and osteopontin decreased. The alkaline phosphatase activity of osteoblasts cultured on PLGA scaffolds is comparable with that from two commercially-available scaffolds - OPLA and collagen scaffolds (Becton-Dickinson (BD) Inc., Franklin Lakes, NJ, USA). Hence, the results suggested that the PLGA scaffolds examined are conducive for promoting osteogenesis.     

Proliferation of chondrocytes on porous poly(DL-lactide)/chitosan scaffolds

Porous poly(DL-lactide)(PDLLA)/chitosan scaffolds with well-controlled pore structures and desirable mechanical characteristics were fabricated via a combination of solvent extraction, phase separation and freeze-drying. These scaffolds were further evaluated for the proliferation of isolated rabbit chondrocytes in vitro for various incubation periods up to 4 weeks in order to finally use them for the cartilage tissue engineering. MTT assay data revealed that the number of cells grown on PDLLA/chitosan scaffolds measurably increased with the weight ratio of the chitosan component and was significantly higher than those collected from pure PDLLA scaffolds for the entire incubation period. Scanning electron microscopy examinations, histological observations and proteoglycan measurements indicated that the resulting PDLLA/chitosan scaffolds exhibited increasing ability to promote the attachment and proliferation of chondrocytes, and also helped seeded chondrocytes spread through the scaffolds and distribute homogeneously inside compared to pure PDLLA scaffolds. Immunohistochemical staining verified that these PDLLA/chitosan scaffolds could preserve the phenotype of chondrocyte and effectively support the production of type II collagen.     

In vivo conjunctival reconstruction using modified PLGA grafts for decreased scar formation and contraction

The in vivo reconstruction of conjunctiva was investigated by using modified poly(lactide-co-glycolide) (PLGA) 50/50 scaffolds. The porous PLGA matrices were prepared by a solvent-casting particulate-leaching method with NaCl, then modified with collagen, hyaluronic acid (HA) or/and human amniotic membrane (AM) component. The growth of corneal epithelial cells and human stromal fibroblasts on the scaffolds was investigated in vitro. All the modified PLGA scaffolds demonstrated enhanced cell adhesion and proliferation as compared to PLGA untreated, and the number of cells proliferated after 1 week was increased in the order of PLGA相似文献   

Responses of mesenchymal stem cell to chitosan-coralline composites microstructured using coralline as gas forming agent

Macroporous composites made of coralline:chitosan with new microstructural features were studied for their scaffolding potential in in vitro bone regeneration. By using different ratios of natural coralline powder, as in situ gas forming agent and reinforcing phase, followed by freeze-drying, scaffolds with controlled porosity and pore structure were prepared and cultured with mesenchymal stem cells (MSCs). Their supportive activity of cellular attachment, proliferation and differentiation were assessed through cell morphology studies, DNA content, alkaline phosphatase (ALP) activity and osteocalcin (OC) release. The coralline scaffolds showed by far the highest evaluation of cell number and ALP activity over all the other chitosan-based scaffolds. They were the only material on which the OC protein was released throughout the study. When used as a component of the chitosan composite scaffolds, these coralline's favourable properties seemed to improve the overall performance of the chitosan. Distinct cell morphology and osteoblastic phenotype expression were observed depending on the coralline-to-chitosan ratios composing the scaffolds. The coralline-chitosan composite scaffolds containing high coralline ratios generally showed higher total cell number, ALP activity and OC protein expression comparing to chitosan scaffolds. The results of this study strongly suggest that coralline:chitosan composite, especially those having a high coralline content, may enhance adhesion, proliferation and osteogenic differentiation of MSCs in comparison with pure chitosan. Coralline:chitosan composites could therefore be used as attractive scaffolds for developing new strategies for in vitro tissue engineering.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering

Unlike braided fabrics, knitted scaffolds have been proven to favor deposition of collagenous connective tissue matrix, which is crucial for tendon/ligament reconstruction. But cell seeding of such scaffolds often requires a gel system, which is unstable in a dynamic situation, especially in the knee joint. This study developed a novel, biodegradable nano-microfibrous polymer scaffold by electrospinning PLGA nanofibers onto a knitted PLGA scaffold in order to provide a large biomimetic surface for cell attachment. Porcine bone marrow stromal cells were seeded onto either the novel scaffolds by pipetting a cell suspension (Group I) or the knitted PLGA scaffolds by immobilizing in fibrin gel (Group II). Cell attachment at 36 hours, cell proliferation and extracellular matrix synthesis at 1 week, and mechanical properties over 2 weeks were investigated. Cell attachment was comparable and cell proliferation was faster in Group I. Moreover, cellular function was more actively exhibited in Group I, as evident by the higher expression of collagen I, decorin, and biglycan genes. Thus, this novel scaffold, facilitating cell seeding and promoting cell proliferation, function, and differentiation, could be applied with promise in tissue engineering of tendon/ligament.