骨髓间质干细胞与纤维蛋白胶生物相容性的实验研究

目的采用体外细胞培养法对纤维蛋白胶的生物相容性进行研究,探讨它作为骨组织工程的载体材料的可行性.方法取2月龄新西兰兔,麻醉后无菌条件下自股骨大转子处抽取骨髓4~6ml, 淋巴细胞分离液离心后取单核细胞层,D-Hanks液洗涤离心2遍,悬浮于含10%FBS的DMEM培养液中,行原代培养.传代的细胞分为2组,一组继续用完全培养基培养,一组用条件培养基培养.分别收集细胞接种于含纤维蛋白胶的培养板中.不含材料的培养板中接种细胞作为对照.于不同时间进行处理,用于相差显微镜与扫描电镜观察,行MTT及碱性磷酸酶的含量测定.结果骨髓间质干细胞可以在纤维蛋白胶表面生长,逐渐粘附.对照组与实验组在细胞光吸收值(OD值)、碱性磷酸酶的含量方面相近,统计学分析无显著差异.结论纤维蛋白胶对兔骨髓间质干细胞的形态学,细胞生长增殖、分化都无影响.具有良好的生物相容性,可作为骨髓间质干细胞的生物载体.     

骨髓间质干细胞与纤维蛋白胶生物相容性的实验研究

目的 采用体外细胞培养法对纤维蛋白胶的生物相容性进行研究，探讨它作为骨组织工程的载体材料的可行性。方法 取2月龄新西兰兔，麻醉后无菌条件下自股骨大转子处抽取骨髓4～6ml，淋巴细胞分离液离心后取单核细胞层，D-Hanks液洗涤离心2遍，悬浮于含10％FBS的DMEM培养液中，行原代培养。传代的细胞分为2组，一组继续用完全培养基培养，一组用条件培养基培养。分别收集细胞接种于含纤维蛋白胶的培养板中。不含材料的培养板中接种细胞作为对照。于不同时间进行处理，用于相差显微镜与扫描电镜观察，行MTT及碱性磷酸酶的含量测定。结果 骨髓间质干细胞可以在纤维蛋白胶表面生长，逐渐粘附。对照组与实验组在细胞光吸收值(OD值)、碱性磷酸酶的含量方面相近，统计学分析无显著差异。结论 纤维蛋白胶对兔骨髓间质干细胞的形态学，细胞生长增殖、分化都无影响。具有良好的生物相容性，可作为骨髓间质干细胞的生物载体。     

Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold

The objectives of this study were (1) to develop a three-dimensional collagen/chitosan/glycosaminoglycan (GAG) scaffold in combination with transforming growth factor-beta1 (TGF-beta 1)-loaded chitosan microspheres, and (2) to evaluate the effect of released TGF-beta 1 on the chondrogenic potential of rabbit chondrocytes in such scaffolds. TGF-beta 1 was loaded into chitosan microspheres using an emulsion-crosslinking method. The controlled release of TGF-beta 1, as measured by enzyme-linked immunosorbent assay (ELISA), was monitored for 7 days. The porous scaffolds containing collagen and chitosan were fabricated by using a freeze drying technique and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) in the presence of chondroitin sulfate (CS), as a GAG component. The TGF-beta 1 microspheres were encapsulated into the scaffold at a concentration of 10 ng TGF-beta 1/scaffold and then chondrocytes were seeded in the scaffold and incubated in vitro for 3 weeks. Both proliferation rate and glycosaminoglycan (GAG) production were significantly higher in the TGF-beta 1 microsphere-incorporated scaffolds than in the control scaffolds without microspheres. Extracellular matrix staining by Safranin O and immunohistochemistry for type II collagen were elevated in the scaffold with TGF-beta 1 microspheres. These results suggest that TGF-beta 1 microspheres when incorporated into a scaffold have the potential to enhance cartilage formation.     

Stem cell based tissue engineering for meniscus repair

Defects of the meniscus greatly alter knee function and predispose the joint to degenerative changes. The purpose of this study was to test a recently developed cell-scaffold combination for the repair of a critical-size defect of the rabbit medial meniscus. A bilateral, complete resection of the pars intermedia of the medial meniscus was performed in 18 New Zealand White rabbits. A hyaluronan/gelatin composite scaffold was implanted into the defect of one knee of 6 rabbits and the contralateral defect was left untreated. Scaffolds loaded with autologous marrow-derived mesenchymal stem cells and cultured in a chondrogenic medium for 14 days were implanted in a second series of 12 rabbits. Empty scaffolds were implanted in the contralateral knees. Meniscii were harvested at 12 weeks. Untreated defects had a muted fibrous healing response. Defects treated with cell-free implants showed also predominantly fibrous tissue whereas fibrocartilage was present in some scaffolds. The cross-sectional width of the repair tissue after treatment with cell-free scaffolds was significantly greater than controls (p < 0.05). Pre-cultured implants integrated with the host tissue and 8 of 11 contained meniscus-like fibrocartilage, compared with 2 of 11 controls (p < 0.03). The mean cross-sectional width of the pre-cultured implant repair tissue was greater than controls (p < 0.004). This study demonstrates the repair of a critical size meniscal defect with a stem cell and scaffold based tissue engineering approach.     

The role of transforming growth factor-beta and bone morphogenetic protein with fibrin glue in healing of bone-tendon junction injury

Bone-tendon junction injuries have poor healing potential. This study evaluated the role of TGF-beta and BMP-2 in a fibrin glue carrier in healing of injuries at bone-tendon junction. Seventy-two skeletally mature male rabbits were divided into 4 groups. The tendo-Achilles was surgically transected at its insertion and reattached with a pullout suture. Group 1 served as a control. In groups 2, 3, and 4, fibrin glue, a mixture of TGF-beta and fibrin glue, and a mixture of BMP-2 and fibrin glue were injected into the bone-tendon junction. The animals were sacrificed at 2, 4 and 8 weeks after surgical procedure. The addition of TGF-beta to fibrin glue did not significantly improve the biomechanical properties of repair tissue. BMP-2 in combination with fibrin glue accelerates healing in a bone-tendon injury and also improves the histological and biomechanical properties of the repair tissue so formed.     

Porous gelatin-chondroitin-hyaluronate tri-copolymer scaffold containing microspheres loaded with TGF-beta1 induces differentiation of mesenchymal stem cells in vivo for enhancing cartilage repair

The aim of the study was to produce a novel porous gelatin-chondroitin-hyaluronate scaffold in combination with a controlled release of transforming growth factor beta1 (TGF-beta1), which induced the differentiation of mesenchymal stem cells (MSCs) in vivo for enhancing cartilage repair. Gelatin microspheres loaded with TGF-beta1 (MS-TGFbeta1) showed a fast release at the initial phase (37.4%), and the ultimate accumulated release was 83.1% by day 18. The autologous MSCs seeded on MS-TGFbeta1/scaffold were implanted to repair full-thickness cartilage defects in rabbits as in vivo differentiation repair group, while MSCs differentiated in vitro were seeded on scaffold without MS-TGFbeta1 to repair the contra lateral cartilage defects (n = 30). Fifteen additional rabbits without treatment for defects were used as control. Histology observation showed that the in vivo differentiation repair group had better chondrocyte morphology, integration, continuous subchondral bone, and much thicker newly formed cartilage layer when compared to in vitro differentiation repair group 12 and 24 weeks, postoperatively. There was a significant difference in histological grading score between these two experimental groups, and both showed much better repair than that of the control. The present study implied that the novel scaffold with MS-TGFbeta1 might serve as a new way to induce the differentiation of MSCs in vivo to enhance the cartilage repair.     

Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge

The aim of this study was to investigate the potential of a composite matrix, containing esterified hyaluronic acid and gelatin, to facilitate the osteochondral differentiation of culture-expanded, bone marrow-derived mesenchymal progenitor cells. The cell loading characteristics and the effects of the matrix on cell differentiation were examined in vitro and in vivo. Empty and cell-loaded composites were cultivated for up to 28 days in a chemically defined medium with or without transforming growth factor-beta1 (TGF-beta1). A type II collagen-rich extracellular matrix was produced by cells loaded in the matrix and cultured in the presence of TGF-beta1. Empty and cell-loaded matrices were also implanted subcutaneously in immunodeficient mice. Three types of implant were used: empty (group I), cell-loaded matrices (Group II), and cell-loaded matrices cultured for 14 days in vitro in defined medium with TGF-beta1 (group III). No osteochondral differentiation was found in implanted empty matrices; however, the matrix supported osteochondrogenic cell differentiation in the cell-loaded implants. Preculture in vitro in a chondrogenic medium increased the percentage of osteochondral tissue found in the constructs after 3 weeks. These results indicate the potential use of this composite matrix for delivery of bone marrow-derived mesenchymal progenitor cells for the repair of chondral and osseous defects. The results also indicate that this composite matrix is useful for in vitro tissue engineering.     

浓缩红骨髓/富血小板纤维蛋白复合载自体骨膜碎片修复下颌骨缺损

背景：富血小板纤维蛋白支架结构有利于红骨髓中间充质干细胞及各种生长因子的生长,促进最终成骨。 目的：探讨浓缩红骨髓/富血小板纤维蛋白复合载自体骨膜碎片支架材料修复兔下颌骨缺损的可行性。 方法：制备新西兰大白兔双侧下颌骨人工制备骨缺损模型,采用自身对照方法,左侧为实验侧,植入自体浓缩红骨髓/富血小板纤维蛋白复合载自体骨膜碎片与纳米羟基磷灰石支架材料;右侧为对照侧,植入自体骨膜碎片复合纳米羟基磷灰石支架材料。术后2,4,8,12周制备组织标本,行大体观察、影像学分析、苏木精-伊红染色、扫描电镜检测。 结果与结论：影像学检查及组织学染色显示,实验侧骨缺损处愈合程度、成骨速度及质量明显优于对照侧;扫描电镜显示实验侧复合材料与组织相容性好,无炎症刺激反应;牙CT分析数据及新骨形成情况分别证明实验侧骨密度CT值显著高于对照侧(P < 0.05),实验侧新生骨面积显著高于对照侧(P < 0.05)。表明浓缩红骨髓/富血小板纤维蛋白复合载自体骨膜碎片支架材料具有明显骨诱导作用,可望成为临床应用中修复颌骨缺损的新型材料。     

Segmental bone regeneration using an rhBMP-2-loaded gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model

We aimed to develop a hybrid scaffold with a porous structure and similar composition as natural bone for the controlled release of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration. We fabricated a gelatin/nanohydroxypatite (nHAP) scaffold by glutaraldehyde chemical cross-linking a gelatin aqueous solution with nHAP granules at a 5:1 ratio (v/w). Then, fibrin glue (FG) mixed with recombinant human BMP-2 (rhBMP-2) was infused into the gelatin/nHAP scaffold and lyophilized to develop an rhBMP-2-loaded gelatin/nHAP/FG scaffold. On scanning electron microscopy, the composite had a 3-D porous structure. The rhBMP-2 release kinetics from the hybrid scaffold was sustained and slow, and release of rhBMP-2 was complete at 40 days. Immunohistochemistry, azo-coupling and alizarin S-red staining were used to study in vitro differentiation of human bone-marrow mesenchymal cells (hBMSCs). Strong positive staining results confirmed that rhBMP-2 released from the scaffold could improve osteocalcin (OCN) and alkaline phosphatase (ALP) expression and calcium deposition formation. RT-PCR results showed significantly high mRNA expression of ALP and OCN in hBM-MSCs cultured on the gelatin/nHAP/FG scaffold with rhBMP-2. DNA assay demonstrated that the scaffold was noncytotoxic and could promote hBMSC proliferation from the components of the hybrid scaffold, not released rhBMP-2. The hybrid scaffolds were then used to repair critical-size segmental bone defects of rabbit radius. Gross specimen, X-ray, bone histomorphology and bone mineral density assay demonstrated that the rhBMP-2-loaded gelatin/nHAP/FG scaffold had good osteogenic capability and could repair the segmental bone defect completely in 12 weeks.     

Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter x 5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host-tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site.     

Repair of diaphyseal bone defects with calcitriol-loaded PLGA scaffolds and marrow stromal cells

Calcitriol (1,25(OH)2D3)-loaded porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds prepared by solvent casting/salt leaching method were used to repair a 1.5 cm diaphyseal segmental bone defect as a fully absorbable osteogenic biomaterial. The in vitro release of sulforhodamine B (SRB) from PLGA scaffold was measured using spectrophotometer, considering SRB as a model drug. The SRB released from SRB-incorporated PLGA scaffold during 3 months was with relatively low initial burst. The calcitriol-loaded PLGA scaffolds with or without marrow stromal cells (MSCs) were implanted in a critical-sized intercalated bone defect in rabbit femur. Defects were assessed by radiographs until 9 weeks. The bony union of the defect was observed only in the calcitriol-loaded groups. RT-PCR results indicated that MSCs, which were seeded into calcitriol-loaded scaffold, expressed an increased level of alkaline phosphatase, osteonectin, and type I collagen mRNA at day 10. After 2 and 4 weeks, the implanted scaffolds were evaluated by histology. New osteoid matrix and direct calcium deposits were more evident in calcitriol/PLGA/MSC group. Three-dimensional computed tomography and frontal tomographic images of repaired femur showed that normal femur anatomy had been restored with cortical bone with no implanted PLGA remnants at 20 weeks. It can be concluded that the porous calcitriol-loaded PLGA scaffold combined with MSCs may be a novel method for repairing the large loaded bone defect.     

Delivery of VEGF using collagen-coated polycaprolactone scaffolds stimulates angiogenesis

Establishing sufficient vascularization in scaffold remains a challenge for tissue-engineering. To improve angiogenesis, we incorporated vascular endothelial growth factor (VEGF) in collagen-coating over the porous polycaprolactone (PCL) scaffolds. The release kinetics of loaded VEGF from collagen-coated PCL (col-PCL) scaffolds was same as from scaffolds without the collagen. The bioactivity of VEGF delivered by the col-PCL scaffolds was confirmed by human umbilical vein endothelial cell (HUVEC) proliferation and chorioallantoic membrane (CAM) assay. The col-PCL scaffolds were implanted subcutaneously in mice for 7 and 14 days. At day 7, vascularization within scaffolds loaded with VEGF was superior to that in the scaffolds without VEGF. However, the vessel connectivity to host circulatory system was incomplete and restricted to the scaffold edges. At day 14, blood vessels in scaffolds reached density similar to the subcutaneous tissue and were perfusable throughout the implant thickness. Prewashing the scaffolds with saline to remove the unbound growth factor decreased the initial burst release and sustained the VEGF-mediated angiogenesis in vivo. In conclusion, our study demonstrates that physically adsorbed VEGF stimulated angiogenesis in collagen-coated PCL scaffolds.     

Soft and hard tissue response to photocrosslinked poly(propylene fumarate) scaffolds in a rabbit model.

The treatment of large cranial defects may be greatly improved by the development of precisely formed bone tissue engineering scaffolds. Such scaffolds could be constructed by using UV laser stereolithography to photocrosslink a linear, biodegradable polymer into a three-dimensional implant. We have previously presented a method to photocrosslink the biodegradable polyester, poly(propylene fumarate) (PPF). To ensure the safety and effectiveness of this technique, the soft and hard tissue response to photocrosslinked PPF scaffolds of different pore morphologies was investigated. Four classes of photocrosslinked PPF scaffolds, constructed with differing porosities (57-75%) and pore sizes (300-500 or 600-800 microm), were implanted both subcutaneously and in 6.3-mm-diameter cranial defects in a rabbit model. The rabbits were sacrificed at 2 and 8 weeks, and the implants were analyzed by light microscopy, histological scoring analysis, and histomorphometric analysis. Results showed the PPF scaffolds elicit a mild tissue response in both soft and hard tissues. Inflammatory cells, vascularization, and connective tissue were observed at 2 weeks; a decrease in inflammatory cell density and a more organized connective tissue were observed at 8 weeks. Scaffold porosity and scaffold pore size were not found to significantly affect the observed tissue response. Evidence of scaffold surface degradation was noted both by histology and histomorphometric analysis. Bone ingrowth in PPF scaffolds implanted into cranial defects was <3% of the defect area. The results indicate that photocrosslinked PPF scaffolds are biocompatible in both soft and hard tissues and thus may be an attractive platform for bone tissue engineering.     

Effect of autologous bone marrow stromal cell seeding and bone morphogenetic protein-2 delivery on ectopic bone formation in a microsphere/poly(propylene fumarate) composite

A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8 microg BMP-2 per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2-loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2-impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8 microg/mg BMP-2-loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2-impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.     

The role of biological extracellular matrix scaffolds in vascularized three-dimensional tissue growth in vivo

An in vivo murine vascularized chamber model has been shown to generate spontaneous angiogenesis and new tissue formation. This experiment aimed to assess the effects of common biological scaffolds on tissue growth in this model. Either laminin-1, type I collagen, fibrin glue, hyaluronan, or sea sponge was inserted into silicone chambers containing the epigastric artery and vein, one end was sealed with adipose tissue and the other with bone wax, then incubated subcutaneously. After 2, 4, or 6 weeks, tissue from chambers containing collagen I, fibrin glue, hyaluronan, or no added scaffold (control) had small amounts of vascularized connective tissue. Chambers containing sea sponge had moderate connective tissue growth together with a mild "foreign body" inflammatory response. Chambers containing laminin-1, at a concentration 10-fold lower than its concentration in Matrigel, resulted in a moderate adipogenic response. In summary, (1) biological hydrogels are resorbed and gradually replaced by vascularized connective tissue; (2) sponge-like matrices with large pores support connective tissue growth within the pores and become encapsulated with granulation tissue; (3) laminin-containing scaffolds facilitate adipogenesis. It is concluded that the nature and chemical composition of the scaffold exerts a significant influence on the amount and type of tissue generated in this in vivo chamber model.     

Repair of calvarial defects with customized tissue-engineered bone grafts I. Evaluation of osteogenesis in a three-dimensional culture system

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.     

壳聚糖-磷酸钙/骨形态发生蛋白2复合骨髓间充质干细胞促进兔脊柱的融合

背景：前期实验已经证明壳聚糖-磷酸钙/骨形态发生蛋白2复合材料能够促进兔脊柱融合。 目的：评价壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子支架材料在兔椎间融合中的应用效果。 方法：制备壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子支架材料,并与大鼠骨髓间充质干细胞在体外构成组织工程骨。摘除40只新西兰大白兔椎间盘,随机分为4组：空白对照组未植入任何材料,对照组植入自体髂骨,支架材料组植入壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子复合材料,实验组植入组织工程骨材料。 结果与结论：术后12周：①X射线片：对照组与实验组椎体融合,两组间融合节段生物力学强度大致相同,生物力学强度高于空白对照组与支架材料组(P < 0.05),且支架材料组高于空白对照组(P < 0.05)。②组织学切片：实验组与对照组有编织骨岛和新生毛细血管生成,支架材料组仅观察到壳聚糖支架网络,空白对照组未发现特殊组织结构。表明壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子复合鼠骨髓间充质干细胞能够明显促进脊柱融合。     

Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vivo

Optimal repair of chondral defects is likely to require both a suitable population of chondrogenic cells and a biodegradable matrix to provide a space-filling structural support during the early stages of cartilage formation. This study examined the ability of chondrocytes to support cartilage formation when incorporated into biodegradable scaffolds constructed from copolymers (PLG) of polylactic acid (PLA) and polyglycolic acid (PGA) and implanted in the calf muscle of nude mice. Scaffolds were fabricated to be more hydrophilic (PLG-H) or were reinforced with 10% PGA fibers (PLG-FR), increasing the stiffness of the implant by 20-fold. Confluent primary cultures of rat costochondral resting zone chondrocytes (RC) were loaded into PLG-H foams and implanted intramuscularly. To determine if growth factor pretreatment could modulate the ability of the cells to form new cartilage, RC cells were pretreated with recombinant human platelet derived growth factor-BB IPDGF-BB) for 4 or 24 h prior to implantation. To assess whether scaffold material properties could affect the ability of chondrogenic cells to form cartilage, RC cells were also loaded into PLG-FR scaffolds. To determine if the scaffolds or treatment with PDGF-BB affected the rate of chondrogenesis, tissue at the implant site was harvested at four and eight weeks post-operatively, fixed, decalcified and embedded in paraffin. Sections were obtained along the transverse plane of the lower leg, stained with haematoxylin and eosin, and then assessed by morphometric analysis for area of cartilage, area of residual implant, and area of fibrous connective tissue formation (fibrosis). Whether or not the cartilage contained hypertrophic cells was also assessed. The amount of residual implant did not change with time in any of the implanted tissues. The area occupied by PLG-FR implants was greater than that occupied by PLG-H implants at both time points. All implants were surrounded by fibrous connective tissue, whether they were seeded with RC cells or not. The amount of fibrosis was reduced at eight weeks for both implant types. When RC cells were present, the amount of fibrosis was less than seen in cell-free scaffolds. Pretreatment with PDGF-BB caused a slightly greater degree of fibrosis at four weeks than was seen if untreated cells were used in the implants. However, at eight weeks, if the cells had been exposed to PDGF-BB for 24 h, fibrosis was comparable to that seen associated with cell-free scaffolds. The cells supported an equivalent area of cartilage formation in both scaffolds. PDGF-BB caused a time-dependent decrease in cartilage formation at four weeks, but at eight weeks, there was a marked increase in cartilage formation in PDGF-BB-treated cells that was greatest in cells exposed for 4 h compared to those exposed for 24 h. Moreover, PDGF-BB decreased the formation of hypertrophic cells. The results indicate that in this model, RC cells produce cartilage; pretreatment of the RC cells with PDGF-BB promotes retention of a hyaline-like chondrogenic phenotype; and the material properties of the implant do not negatively impact on the ability of the cells to support chondrogenesis.     

Fibrin glue mixed with gelatin/hyaluronic acid/chondroitin-6-sulfate tri-copolymer for articular cartilage tissue engineering: the results of real-time polymerase chain reaction

Autologous fibrin glue has been demonstrated as a potential scaffold with very good biocompatibility for neocartilage formation. However, fibrin glue has been reported not to provide enough mechanical strength, but with many growth factors to interfere the tissue growth. Gelatin/hyaluronic acid/chondroitin-6-sulfate (GHC6S) tri-copolymer sponge has been prepared as scaffold for cartilage tissue engineering and showed very good results, but problems of cell seeding and cell distribution troubled the researchers. In this study, GHC6S particles would be added into the fibrin glue to provide better mechanical strength, better cell distribution, and easier cell seeding, which would be expected to improve cartilage regeneration in vitro. Porcine cryo-precipitated fibrinogen and thrombin prepared from prothrombin activated by 10% CaCl(2) solution were used in two groups. One is the fibrin glue group in which porcine chondrocytes were mixed with thrombin-fibrinogen solution, which was then converted into fibrin glue. The other is GHC6S-fibrin glue in which GHC6S particles were added into the thrombin-fibrinogen solution with porcine chondrocytes. After culturing for 1-2 weeks, the chondrocytes cultured in GHC6S-fibrin glue showed a round shape with distinct lacuna structure and showed positive in S-100 protein immunohistochemical stain. The related gene expressions of tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-2, MT1-MMP, aggrecan, decorin, type I, II, X collagen, interleukin-1 beta, transforming growth factor-beta 1 (TGF-beta1), and Fas-associating death domain were checked by real-time PCR. The results indicated that the chondrocytes cultured in GHC6S-fibrin glue would effectively promote extracellular matrix (ECM) secretion and inhibit ECM degradation. The evidence could support that GHC6S-fibrin glue would be a promising scaffold for articular cartilage tissue engineering.     

丝素蛋白-壳聚糖支架复合骨髓间充质干细胞体内构建组织工程化软骨的生物相容性

文题释义： 生物相容性：是指生命体组织对非活性材料产生的一种性能,一般是指材料与宿主之间的相容性,包括组织相容性和血液相容性。 检测相容性的方法：是将支架材料与种子细胞在体外共培养,检测支架毒性、细胞活性、细胞增殖及细胞与支架的黏附情况等指标,该方法具有客观性强、可重复性强、影响因素相对简单及敏感性高等特点。 背景：课题组前期的研究中发现,丝素蛋白-壳聚糖支架材料复合诱导后骨髓间充质干细胞在兔体内能修复缺损的软骨组织,但对于该组织工程化软骨组织的生物相容性还未进一步研究。 目的：研究丝素蛋白-壳聚糖支架材料复合骨髓间充质干细胞在体内构建组织工程化软骨的生物相容性。 方法：使用丝素蛋白-壳聚糖按1∶1比例混合制备三维支架材料,提取兔骨髓间充质干细胞,将诱导后的骨髓间充质干细胞与丝素蛋白-壳聚糖支架构建修复体,再将修复体移植到兔关节软骨缺损模型中修复软骨组织。实验分为3组,实验组植入诱导后骨髓间充质干细胞+丝素蛋白-壳聚糖支架,对照组植入丝素蛋白-壳聚糖支架干预,空白组未植入修复体。 结果与结论：①实验成功制备丝素蛋白-壳聚糖三维支架材料及提取骨髓间充质干细胞,并构建软骨缺损的修复体,将修复体植入兔体内能成功修复缺损的软骨组织;②建模后2,4,8,12周,3组血常规、降钙素原、血沉、C-反应蛋白结果提示无明显的全身感染征象,3组血常规及肝肾功能各时间段比较差异无显著性意义(P > 0.05);③一般观察、苏木精-伊红染色及扫描电镜观察：建模后12周,相比其他两组,实验组软骨缺损已修复,支架材料已吸收,修复组织周围未见炎性细胞,修复组织已正常组织整合良好;④结果证实,丝素蛋白-壳聚糖支架复合骨髓间充质干细胞在体内构建的组织工程化软骨具有良好的生物相容性。 ORCID: 0000-0002-8139-1175(佘荣峰) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程