Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle

OBJECTIVE: Adult articular cartilage shows a limited intrinsic repair response to traumatic injury. To regenerate damaged cartilage, cell-assisted repair is thus viewed as a promising therapy, despite being limited by the lack of a suitable technique to deliver and retain chondrogenic cells at the defect site. DESIGN: We have developed a cytocompatible chitosan solution that is space-filling, gels within minutes, and adheres to cartilage and bone in situ. This unique combination of properties suggested significant potential for its use as an arthroscopically injectable vehicle for cell-assisted cartilage repair. The primary goal of this study was to assess the ability of this polymer system, when loaded with primary articular chondrocytes, to support cartilage formation in vitro and in vivo. The chitosan gel was cultured in vitro, with and without chondrocytes, as well as injected subcutaneously in nude mice to form subcutaneous dorsal implants. In vitro and in vivo constructs were collectively analyzed histologically, for chondrocyte mRNA and protein expression, for biochemical levels of glycosaminoglycan, collagen, and DNA, and for mechanical properties. RESULTS: Resulting tissue constructs revealed histochemical, biochemical and mechanical properties comparable to those observed in vitro for primary chondrocytes cultured in 2% agarose. Moreover, the gel was retained after injection into a surgically prepared, rabbit full-thickness chondral defect after 1 day in vivo, and in rabbit osteochondral defects, up to 1 week. CONCLUSIONS: The in situ-gelling chitosan solution described here can support in vitro and in vivo accumulation of cartilage matrix by primary chondrocytes, while persisting in osteochondral defects at least 1 week in vivo.     

Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads

OBJECTIVE: For autologous chondrocyte transplantation (ACT) chondrocytes are expanded in vitro. During expansion these cells may dedifferentiate. This change in phenotype is characterized by a raised expression of type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of hyaline cartilage, we investigated if the growth factor bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures and in three-dimensional culture systems. DESIGN: Chondrocytes from articular knee cartilage of 11 individuals (average age: 39.8 years) with no history of joint disease were isolated and seeded either in monolayer cultures or embedded in alginate beads in presence or absence of human recombinant BMP-2 (hr-BMP-2). Then, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA. RESULTS: Addition of BMP-2 to chondrocytes expanded in two-dimensional (2D) cultures during the first subculture (P1) had no effect on mRNA amounts encoding type II collagen and interleukin-1beta (IL-1beta). In contrast, seeding chondrocytes in three-dimensional (3D) alginate cultures raised type II collagen expression significantly and addition of BMP-2 enhanced this effect. CONCLUSIONS: We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system.     

Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis

OBJECTIVE: If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells. METHODS: Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2-8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker 'activated' caspase-3 in monolayer or secondary alginate cultures. RESULTS: The synthesis of cartilage-specific type-II collagen, alpha 3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis. CONCLUSION: We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.     

Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin.

We report a novel method to culture chick embryo osteoblasts in vitro. Primary cells were grown from explants of calvaria and then cultured within alginate polymer beads. Enriched cultures of primary osteoblasts were obtained because these cells grow readily within alginate beads but other cell types present in the initial outgrowth from calvarial fragments, such as fibroblasts, do not. A reproducible bone cell phenotype was observed in calvarial cells cultured in the alginate polymer for as long as 8 months. Alginate is a uronic acid monomer that reversibly polymerizes based on the presence or absence of divalent cations. Osteoblasts derived from the alginate beads elaborated and mineralized an extracellular matrix in vitro that contained fibronectin, type III collagen, and type I collagen. The synthesis and deposition of these matrix molecules was also demonstrated in the chick embryo calvaria in vivo. Together, these in vitro and in vivo observations provide the first evidence that type III collagen and fibronectin colocalize with type I collagen during the development of avian membranous bone. They also indicate that the phenotype of chick embryo osteoblasts can be expanded to include the synthesis of fibronectin and type III collagen.     

Characterization of human nasal septal chondrocytes cultured in alginate

BACKGROUND: After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. STUDY DESIGN: Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. RESULTS: Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. CONCLUSIONS: Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.     

Culture of human septal chondrocytes in a rotary bioreactor

Objectives (1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture. Study Design Prospective, basic science. Setting Research laboratory. Methods Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured. Results Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P < .01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0. Conclusion Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Response of human chondrocytes prepared for autologous implantation to growth factors

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.     

经碱性成纤维细胞生长因子修饰骨髓基质干细胞注射式修复全层软骨缺损

目的 探讨经碱性成纤维细胞生长因子(bFGF)修饰的骨髓基质干细胞(BMSCs)注射式修复全层软骨缺损的可行性.方法 体外培养家兔自体骨髓基质干细胞,并以bFGF处理修饰细胞,免疫组织化学Ⅱ型胶原蛋白表达、逆转录-聚合酶链反应(RT-PCR)检测蛋白聚糖表达.将其与藻酸钙凝胶支架复合注射式植入兔股骨髁全层软骨缺损处,同时设立凝胶支架对照组和空白对照组.术后8周取材观察修复效果,并行苏木素-伊红(HE)、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色检测;透射电镜观察修复组织的微观结构.结果 BMSCs的细胞群体倍增时间(PDT)为33.8 h,经bFGF处理的BMSCs可检测到Ⅱ型胶原和蛋白聚糖的表达.至术后8周可见质硬的类白色修复组织完全充填软骨缺损处,组织学检查可见大量软骨样细胞分布于深染的细胞外基质中,检测到Ⅱ型胶原的表达.透射电镜可见丰富的细胞器和胞外基质.凝胶支架对照组和空白对照组仅有部分质软组织充填缺损处,未检测到Ⅱ型胶原的表达.结论 经hFGF修饰的自体骨髓基质干细胞藻酸钙凝胶复合物可用于修复全层软骨缺损.     

Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure

One of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20%. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20% O2 (= 20.26 kPa PO2) + 5% CO2 + 75% N2; 2.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2; 3.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three-dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20% oxygen. With 5% oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5% oxygen lead to a decreased collagen type II-production compared to cells subjected to 5% oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90% after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.     

In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues

Bone marrow stromal cells, progenitor cells involved in repair of bone and cartilage, can potentially provide a source for autologous skeletal tissue engineering. We investigated which factors were required to induce in vitro differentiation of avian bone marrow stromal cells into three-dimensional cartilaginous and bone-like tissues. Bone marrow stromal cells from embryonic chicks were expanded in monolayers, seeded onto biodegradable polyglycolic acid scaffolds, and cultured for 4 weeks in orbitally mixed Petri dishes. Cell-polymer constructs developed an organized extracellular matrix containing glycosaminoglycans and collagen, whereas control bone marrow stromal cell pellet cultures were smaller and consisted predominantly of fibrous tissue. Bone marrow stromal cells expanded with fibroblast growth factor-2 and seeded onto polymer scaffolds formed highly homogeneous three-dimensional tissues that contained cartilage-specific molecular markers and had biochemical compositions comparable with avian epiphyseal cartilage. When cell-polymer constructs were cultured in the presence of beta-glycerophosphate and dexamethasone, the extracellular matrix mineralized and bone-specific proteins were expressed. Our work shows that cell expansion in the presence of fibroblast growth factor-2 and cultivation on a three-dimensional polymer scaffold allows differentiation of chick bone marrow stromal cells into three-dimensional cartilaginous tissues. In the in vitro system studied, the same population could be selectively induced to regenerate either cartilaginous or bonelike tissue.     

Use of autologous tissue engineered skin to treat porcine full-thickness skin defects

Iinn irteiaceln ta uyteoagrrsa,ft sk iann dgra fatlilnoggr hafats e pvroelvpaedra tfiroonms th toebiosynthetic and tissue-engineered living skinreplacements.The production of bioengineered humantissues has led to fascinating diversity of medicalapplications,notably for permanent burn woundcoverage.1For a few decades,various skin substituteshave been introduced for extensive full-thickenss burnmanagement,and also provided new approaches forreconstructive surgery.2-7In addition,allografting ofcu…     

大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架构建组织工程软骨

目的 探讨大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架的优越性.方法 选用6周龄健康Wistar大鼠,分离出脂肪干细胞后行体外培养.将Ⅰ型胶原溶液与壳聚糖溶液混合后冷冻干燥,交联硫酸软骨素后再冷冻干燥得到复合三维支架,检测支架的孔径值、含水量及孔隙率.将接种的脂肪干细胞消化后分别接种到平面、微球和支架,软骨方向诱导培养.MTT检测细胞增殖情况,3周后倒置显微镜及扫描电镜观察细胞形态及在支架上的生长及黏附情况,并分析成软骨分化的情况.结果 5 d后MTT检测显示三维支架组及微球组细胞增殖速度较平面组快;三维支架组14 d后仍有细胞增殖.组织学分析显示细胞在支架上密集重叠生长,内层仍有残留支架结构.Ⅱ型胶原免疫组化检测结果显示,三维支架组及微球组表达呈强阳性,而平面组表达呈弱阳性.RT-PCR结果显示各组均有软骨特异性mRNA的表达.但平面组一直表达X型胶原,微球组培养至21 d时也表达X型胶原,而三维支架组则一直未表达.结论 复合胶原-壳聚糖-硫酸软骨素三维支架能促进细胞的增殖、分化,并能更好地维持软骨细胞的表型,可以作为组织工程构建软骨的最佳选择.     

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self‐assembling peptide hydrogel

Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self‐assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self‐assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the absence of the chondrogenic cytokine transforming growth factor beta (TGFβ). Positive controls consisted of undifferentiated MSCs seeded into agarose and cultured in medium containing TGFβ. Three days of preconditioning was sufficient to produce chondrogenic MSCs that accumulated ～75% more cartilaginous extracellular matrix than positive controls by day 17. However, gene expression of type X collagen was ～65‐fold higher than positive controls, which was attributed to the absence of TGFβ. Potential induction of immunogenicity with preconditioning culture was indicated by expression of major histocompatibility complex class II (MHCII), which was nearly absence in undifferentiated MSCs, and ～7% positive for preconditioned cells. These data demonstrate the potential to generate chondrogenic MSCs with days of self‐assembling peptide hydrogel, and the ability to readily recover an individual cell suspension that is suited for injectable therapies. However, continued exposure to TGFβ may be necessary to prevent hypertrophy indicated by type X collagen expression, while immunogenicity may be a concern for allogeneic applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1368–1375, 2019.     

A novel exogenous concentration-gradient collagen scaffold augments full-thickness articular cartilage repair

OBJECTIVES: A collagen scaffold has been long used in order to enhance the regeneration of articular cartilage. In the present study, we investigate the effectiveness of a concentration-gradient (CG) collagen that is designed to recruit efficiently the mesenchymal stem cells (MSCs) to the central region of the full-thickness cartilage defects via haptotaxis. METHODS: The present study used Cellmatrix((R)) (0.3% type I collagen; Nitta gelatin, Osaka, Japan) as the collagen material. We prepared 33%CG collagen gel and 50%CG collagen gel. No gradient collagen gel served as negative control. Full-thickness cartilage defects were created at the patella groove of the rabbit knee, to which the three different collagen gels were transplanted. Bromodeoxyuridine (BrdU) positive, proliferating cells were enumerated and localized, whereas the histological grading score for cartilage regeneration was counted. The expression of type I and type II collagens was evaluated by immunohistochemistry. We also confirmed that the MSCs migrate toward the collagen substrate of higher concentration in a stringently in vitro haptotactic manner. RESULTS: Enumeration of the BrdU-positive cells demonstrated that 33%CG collagen gel recruited a significantly larger number of proliferating cells to the central region of the cartilage defect. The histological grading score for the regenerated cartilage treated with 33%CG collagen gel was superior to the other groups. CONCLUSIONS: CG collagen scaffold recruits effectively the MSCs to the center of full-thickness cartilage defect and enhances regeneration of the full-thickness cartilage defect.     

兔脊索细胞体外培养及生物学特点观察

目的建立体外兔椎间盘脊索细胞藻酸盐凝胶培养模型,观察脊索细胞形态及生物学特点。方法采用胶原酶消化法及Percoll不连续密度梯度沉淀法体外分离收集原代椎间盘脊索细胞,于1.2%藻酸盐凝胶(低密度)中培养。倒置相差显微镜下观察细胞形态,经Ⅱ型胶原免疫荧光染色对细胞表型初步鉴定,并分别以细胞增殖和细胞毒性试剂-8(CCK-8)检测细胞在藻酸盐凝胶中的存活和增殖能力。结果成功分离获得原代椎间盘脊索细胞,可稳定表达Ⅱ型胶原。原代脊索细胞在藻酸盐凝胶中生长良好,但增殖缓慢。结论初步了解兔椎间盘脊索细胞体外生物学特性,为椎间盘退变机制及组织工程学髓核种子细胞的研究提供一定的实验依据。