Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28?days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p?<?0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.     

Shape-memory porous alginate scaffolds for regeneration of the annulus fibrosus: Effect of TGF-β3 supplementation and oxygen culture conditions

Disc herniation as a result of degenerative or traumatic injury is believed to be the primary instigator of low back pain. At present there is a lack of viable treatment options to repair damaged annulus fibrosus (AF) tissue. Developing alternative strategies to fill and repair ruptured AF tissue is a key challenge. In this work we developed a porous alginate scaffold with shape-memory properties which can be delivered using minimally invasive approaches and recover its original geometry once hydrated. Covalently cross-linked alginate hydrogels were created using carbodiimide chemistry, followed by a freeze-drying step to impart porosity and create porous scaffolds. Results showed that porous alginate scaffolds exhibited shape-memory recovery and mechanical behaviour that could be modulated depending on the cross-linker concentrations. The scaffold can be repeatedly compressed and expanded, which provides the potential to deliver the biomaterial directly to the damaged area of the AF tissue. In vitro experiments demonstrated that scaffolds were cytocompatible and supported cell seeding, penetration and proliferation under intervertebral-disc-like microenvironmental conditions (low glucose media and low oxygen concentration). Extracellular matrix (ECM) was secreted by AF cells with TGF-β3 stimulation and after 21 days had filled the porous scaffold network. This biological matrix was rich in sulfated glycosaminoglycan and collagen type I, which are the main compounds of native AF tissue. Successful ECM deposition was also confirmed by the increase in the peak stress of the scaffold. However, the immaturity of the matrix network after only 21 days of in vitro culture was not sufficient to attain native AF tissue mechanical properties. The ability to deliver porous scaffolds using minimal invasive approaches that can potentially promote the regeneration of AF defects provides an exciting new avenue for disc repair.     

Developing an alginate/chitosan hybrid fiber scaffold for annulus fibrosus cells

A fibrous scaffold made of alginate or alginate/chitosan was fabricated for annulus fibrosus (AF) cell culture using a wet-spinning and lyophilization technique. The scaffolds were evaluated using several in vitro tests. Scanning electron microscopy showed the scaffold fibers generally aligned in one direction with individual fiber diameters varying between 40-100 microm. The alginate/chitosan hybrid scaffold exhibited a slower degradation rate, while both scaffold types did not display any cyto-toxicity to 3T3 fibroblasts and could maintain canine AF cell growth. The AF cells retained their spherical shape within the fibrous scaffold at the beginning of the culture period and formed into cell clusters at later times. Specific extracellular matrix molecules, including collagen I, collagen II, and aggrecan, could be detected in the AF cell clusters. These results demonstrate the feasibility of using this hybrid alginate/chitosan scaffold for AF cell culture, and the potential application for intervertebral disc tissue engineering.     

Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability,morphology, and function

Tissue engineering with three-dimensional biomaterials represents a promising approach for developing hepatic tissue to replace the function of a failing liver. Herein, we address cell seeding and distribution within porous alginate scaffolds, which represent a new type of porous biomaterial for tissue engineering. The hydrophilic nature of the alginate scaffold as well as its pore structure and interconnectivity enabled the efficient seeding of hepatocytes into the scaffolds, that is, 70-90% of the initial cells depending on the seeding method. Utilization of centrifugal force during seeding enhanced cell distribution in the porous scaffolds, consequently enabling the seeding of concentrated cell suspensions (>1 x 10(7) cells/mL). Cell density in scaffolds affected hepatocyte viability as judged by MTT assay. At a cell density of 0.28 x 10(6) cells/cm3 scaffold, the number of viable hepatocytes decreased to 33% of its initial value within 7 days, whereas at the denser cultures, 5.7 x 10(6) cells/cm3 scaffold and higher, the cells maintained higher viability while forming a network of connecting spheroids. In the high-density cellular constructs, hepatocellular functions such as albumin and urea secretion, and detoxification (cytochrome P-450 and phase II conjugating enzyme activities), remained high during the 7-day culture. Collectively, the results of the present study highlight the importance of cell density on the hepatocellular functions of three-dimensional hepatocyte constructs as well as the advantages of alginate matrices as scaffoldings.     

Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering

To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.     

Effect of 3D-microstructure of bioabsorbable PGA:TMC scaffolds on the growth of chondrogenic cells

Various biomaterial scaffolds have been investigated for cartilage tissue engineering, although little attention has been paid to the effect of scaffold microstructure on tissue growth. Non-woven, fibrous, bioabsorbable scaffolds constructed from a copolymer of glycolide and trimethylene carbonate with varying levels of porosity and pore size were seeded with mesenchymal stroma cells with a chondrogenic lineage. Scaffolds and media were evaluated for both cell and extracellular matrix organization and content after up to 28 days of culture in a spinner flask. Analysis of DNA and glycosaminoglycan contents showed that the most porous of the three scaffold types, with a porosity of 81% and a porometry determined mean flow pore diameter of 54 microm, supported the most rapid proliferation of cells and accumulation of extracellular matrix. Analysis of the high porosity scaffold system, using Western Blot and immunohistochemistry confirmed the presence of collagen type II and absence of collagen type I, and demonstrated cells with a chondrocyte morphology with aggrecan and collagen II accumulation attached to the scaffolds. It was concluded that the 3D-microstructural characteristics of the scaffold (interconnecting porosity and pore size) play an important role in proliferation and phenotype of chondrogenic cells and accumulation of extracellular matrix molecules.     

Rat costochondral cell characteristics on poly (L-lactide-co-epsilon-caprolactone) scaffolds

This study was designed to examine the adhesion, proliferation, and morphology of chondrocytes on new scaffolds; and to examine these cells histologically for the ability of the chondrocytes to maintain chondrogenic properties after subcutaneous implantation into nude mice. Both 75:25 poly (L-lactide-co-epsilon-caprolactone) (75PLC) and 50:50 poly (L-lactide-co-epsilon-capro-lactone) scaffold (50PLC) were tested as a scaffold for rat costochondral resting zone chondrocytes in comparison with a type I collagen sponge scaffold (collagen scaffold). Both of the poly (L-lactide-co-epsilon-caprolactone) scaffolds (75PLC and 50PLC) were coated with type I collagen solution and the effects of the collagen coat (hybrid-PLC) were also examined. The hybrid-75PLC bound the same number of cells as the collagen scaffold, whereas the 75PLC and the 50PLC bound 60% and 50% fewer cells than the collagen scaffold, respectively. The cell growth on the scaffolds progressed with culture time in all scaffolds. Cell morphology was assessed by scanning electron microscopy for differences in the structure of cellular interaction. Chondrocytes on every scaffold maintained a spherical shape. The hybrid-PLCs were superior to the PLCs with respect to the number of cells attached. The PLCs had an advantageous degradation characteristic in that they retained their original shape better than the collagen scaffold. Additionally, in the PLCs seeded, the cells retained their integrity 4 weeks after implantation, although the volume of collagen scaffold decreased by 50%.     

Improving the cell distribution in collagen-coated poly-caprolactone knittings

Adequate cellular in-growth into biomaterials is one of the fundamental requirements of scaffolds used in regenerative medicine. Type I collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support can be produced. However, in general, adequate cell in-growth and cell seeding has been suboptimal. In this study we prepared collagen scaffolds of different collagen densities and investigated the cellular distribution. We also prepared a hybrid polymer-collagen scaffold to achieve an optimal cellular distribution as well as sufficient mechanical strength. Collagen scaffolds [ranging from 0.3% to 0.8% (w/v)] with and without a mechanically stable polymer knitting [poly-caprolactone (PCL)] were prepared. The porous structure of collagen scaffolds was characterized using scanning electron microscopy and hematoxylin-eosin staining. The mechanical strength of hybrid scaffolds (collagen with or without PCL) was determined using tensile strength analysis. Cellular in-growth and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and human urothelium seeding. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a hybrid scaffold prepared from 0.4% collagen strengthened with knitting achieved the best cellular distribution.     

Enhancement of viability of muscle precursor cells on 3D scaffold in a perfusion bioreactor

The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.     

Variations in chondrogenesis of human bone marrow-derived mesenchymal stem cells in fibrin/alginate blended hydrogels

Fibrin and alginate hydrogels have been widely used to support chondrogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs) for articular cartilage and fibrocartilage tissue engineering, with each material offering distinct advantages and disadvantages. Attempting to produce a gel scaffold exhibiting beneficial characteristics of both materials, we fabricated fibrin/alginate blended hydrogels at various blend ratios and evaluated the gel morphology, mechanical properties and their support for BM-MSC chondrogenesis. Results show that when the fibrin/alginate ratio decreased, the fibrin architecture transitioned from uniform to interconnected fibrous and finally to disconnected islands against an alginate background, with opposing trends in the alginate architecture. Fibrin maintained gel extensibility and promoted cell proliferation, while alginate improved the gel biostability and better supported glycosaminoglycan and collagen II production and chondrogenic gene expression. Blended gels had physical and biological characteristics intermediate between fibrin and alginate. Of the blends examined, FA 40:8 (40mgml(-1) fibrinogen blended with 8mgml(-1) alginate) was found to be the most appropriate group for future studies on tension-driven BM-MSC fibrochondrogenesis. As BM-MSC differentiation appeared to vary between fibrin and alginate regions of blended scaffolds, this study also highlighted the potential to develop spatially heterogeneous tissues through manipulating the heterogeneity of scaffold composition.     

Porous alginate/hydroxyapatite composite scaffolds for bone tissue engineering: preparation, characterization, and in vitro studies

In this study a series of alginate/hydroxyapatite (HAP) composite scaffolds was prepared by phase separation. HAP was incorporated into the alginate gel solution to improve both the mechanical and cell-attachment properties of the scaffolds. These scaffolds had a well-interconnected porous structure with an average pore size of 150 microm and over 82% porosity. The alginate/HAP scaffold prepared at -40 degrees C with a 50% HAP content showed the best mechanical properties. The morphology of scaffolds could be manipulated by tuning the quenching temperature during the preparation. The dissolution of alginate/HAP composite scaffolds could be slowed by the pretreating them by immersion in 1.0 M CaCl(2) solution. The rat osteosarcoma UMR106 cells, an osteoblastic cell line, seeded in the scaffolds, displayed better cell attachment to the 75/25 and 50/50 alginate/HAP composite scaffolds than to the pure alginate scaffold. The natural polymeric sponges that fabricated in this study may be a promising approach for tissue-engineering applications.     

Engineered channels enhance cellular density in perfused scaffolds

Scaffold-based tissue engineering provides cells with an engineered matrix to enhance and direct cell attachment, proliferation and differentiation. One critical limitation to current tissue engineering approaches is the inability to create densely populated constructs thicker than a few 100 μm. We hypothesized that development of porous, channeled scaffolds would increase cell density and uniformity of their spatial distribution through scaffold channel perfusion. Patterned polyurethane sheets were fabricated using a sprayed phase separation technique and laminated together to form 1.5 mm thick channeled scaffolds. Hydraulic permeability testing confirmed the presence of functional channels throughout the multilaminate construct. A continuous flow bioreactor was used to perfuse the construct with medium during the culture period. Cross-sectional cell densities and spatial uniformities were measured in channeled and nonchanneled scaffolds under different seeding and culture conditions. Channeled scaffolds were found to have higher densities of human mesenchymal stem cells than nonchanneled samples. Perfused scaffolds had more uniform spatial distribution of cells within the scaffold compared to statically cultured scaffolds. In conclusion, we have shown the channeled scaffolds to be a promising approach toward creating thick tissue-engineered constructs.     

Chondrogenesis from human placenta-derived mesenchymal stem cells in three-dimensional scaffolds for cartilage tissue engineering

Human placenta-derived mesenchymal stem cells (hPMSCs) represent a promising source of stem cells. The application of hPMSCs in cartilage tissue engineering, however, was less reported. In this study, hPMSCs were grown in a three-dimensional (3D) environment for cartilage tissue formation in vitro. To select proper scaffolds for 3D culture of mesenchymal stem cells (MSCs), rat adipose-derived MSCs were initially employed to optimize the composition and condition of the 3D environment. The suitability of a poly(D,L-lactide-co-glycolide) (PLGA) precision scaffold previously developed for seeding and culture of primary chondrocytes was tested for MSCs. It was established that MSCs had to be embedded in alginate gel before seeded in the PLGA precision scaffold for cartilage-like tissue formation. The inclusion of nano-sized calcium-deficient hydroxyapatite (nCDHA) and/or a recombinant protein containing arginine-glycine-aspartate (RGD) into the alginate gel enhanced the chondrogenesis for both rat adipose-derived MSCs and hPMSCs. The amount of extracellular matrix such as glycosaminoglycan and type II collagen accumulated during a period of 21 days was found to be the greatest for hPMSCs embedded in the alginate/nCDHA/RGD gel and injected and cultivated in the precision scaffold. Also, histological analyses revealed the lacunae formation and extracellular matrix production from the seeded hPMSCs. Comparing human bone marrow-derived MSCs (hBMSCs) and hPMSCs grown in the previous composite scaffolds, the secretion of glycosaminoglycan was twice as higher for hPMSCs as that for hBMSCs. It was concluded that the alginate/nCDHA/RGD mixed gel in the aforementioned system could provide a 3D environment for the chondrogenesis of hPMSCs, and the PLGA precision scaffold could provide the dimensional stability of the whole construct. This study also suggested that hPMSCs, when grown in a suitable scaffold, may be a good source of stem cells for building up the tissue-engineered cartilage.     

Self-assembled octapeptide scaffolds for in vitro chondrocyte culture

Nature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. Molecular self-assembly is a simple and efficient way to fabricate complex nanostructures such as hydrogels. We have recently investigated the gelation properties of a series of ionic-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptide, FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine). This peptide was shown to self-assemble in solution and form β-sheet-rich nanofibres which, above a critical gelation concentration, entangle to form a self-supporting hydrogel. The fibre morphology of the hydrogel was analysed using transmission electron microscopy and cryo-scanning electron microscopy illustrating a dense fibrillar network of nanometer size fibres. Oscillatory rheology results show that the hydrogel possesses visco-elastic properties. Bovine chondrocytes were used to assess the biocompatibility of the scaffolds over 21 days under two-dimensional (2-D) and three-dimensional (3-D) cell culture conditions, particularly looking at cell morphology, proliferation and matrix deposition. 2-D culture resulted in cell viability and collagen type I deposition. In 3-D culture the mechanically stable gel was shown to support the viability of cells, the retention of cell morphology and collagen type II deposition. Subsequently the scaffold may serve as a template for cartilage tissue engineering.     

Evaluation of different scaffolds for BMP-2 genetic orthopedic tissue engineering

To better understand the effects of scaffold materials for bone morphogenetic protein 2 (BMP-2) genetic tissue engineering in vivo, several gels, including alginate, collagen, agarose, hyaluronate, fibrin, or Pluronic, were mixed with adenovirus-mediated human BMP-2 gene (Adv-hBMP-2) transduced bone marrow stromal cells (BMSCs) and injected into the muscles of athymic mice to evaluate the resulting osteogenesis and chondrogenesis. These gel and gene-transduced BMSC mixtures were also loaded onto beta-TCP/HAP biphasic calcined bone (BCB) and observed under scanning electron microscopy (SEM). In addition, these composite scaffolds were implanted into the subcutaneous site of athymic mice to construct tissue-engineered bone. After injection, collagen, hyaluronate, or alginate gel mixed with gene-transduced BMSCs induced more bone formation than a cell suspension in alpha-MEM. The agarose-gene-transduced BMSC gel was found to contain much more hyaline cartilage. SEM showed the BMSCs could survive in alginate, agarose, and collagen gel in vitro for up to 8 d. After implantation of tissue-engineered bone, the alginate, collagen, and agarose gel could promote new bone formation within a BCB in vivo. Little or no bone formed after injection of fibrin or Pluronic gel mixed with BMSCs or implantation with BCB. These findings help to elucidate the effects of various scaffold materials for future research in orthopedic tissue engineering using BMP-2 transduced cells.     

Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers

In this study, we successfully developed three-dimensional scaffolds fabricated from the chitosan-based hyaluronic acid hybrid polymer fibers, which can control the porous structure. To determine the adequate pore size for enhancing the chondrogenesis of cultured cells, we compared the behaviors of rabbit chondrocytes in scaffolds comprising different pore sizes (100, 200, and 400 microm pore size). Regarding the cell proliferation, there was no significant difference among the three groups. On the other hand, glycosaminoglycan contents in the 400 microm group significantly increased during the culture period, compared with those in the other groups. The ratio of type II to type I collagen mRNA level was also significantly higher in the 400 microm group than in the other groups. These results indicate that our scaffold with 400 microm pore size significantly enhances the extracellular matrix synthesis by chondrocytes. Additionally, the current scaffolds showed high mechanical properties, compared with liquid and gel materials. The data derived from this study suggest great promise for the future of a novel fabricated material with relatively large pore size as a scaffold for cartilage regeneration. The biological and mechanical advantages presented here will make it possible to apply our scaffold to relatively wide cartilaginous lesions.     

Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties

Alginate gels have been used in both drug delivery and cell encapsulation applications in the bead form usually produced by dripping alginate solution into a CaCl2 bath. The major disadvantages to these systems are that the gelation rate is hard to control; the resulting structure is not uniform; and mechanically strong and complex-shaped 3-D structures are difficult to achieve. In this work controlled gelation rate was achieved with CaCO3-GDL and CaSO4-CaCO3-GDL systems, and homogeneous alginate gels were formulated as scaffolds with defined dimensions for tissue engineering applications. Gelation rate increased with increasing total calcium content, increasing proportion of CaSO4, increasing temperature and decreasing alginate concentration. Mechanical properties of the alginate gels were controlled by the compositional variables. Slower gelation systems generate more uniform and mechanically stronger gels than faster gelation systems. The compressive modulus and strength increased with alginate concentration, total calcium content, molecular weight and guluronic acid (G) content of the alginate. MC3T3-E1 osteoblastic cells were uniformly incorporated in the alginate gels and cultured in vitro. These results demonstrated how alginate gel and gel/cell systems could be formulated with controlled structure, gelation rate, and mechanical properties for tissue engineering and other biomedical applications.     

聚己二酸丁二醇酯-对苯二甲酸丁二醇酯/Ⅰ型胶原取向纤维促进前交叉韧带断裂后腱-骨愈合

背景:材料表面的微/纳米结构对细胞的行为具有调控作用,肌腱组织主要由平行的胶原纤维组成,因此取向纤维结构对腱-骨愈合具有一定的促进作用.目的:探索聚己二酸丁二醇酯-对苯二甲酸丁二醇酯[poly(butylene adipate-co-terephthalate),PBAT]/Ⅰ型胶原取向纤维支架的生物相容性和成骨诱导活...     

Atelocollagen for culture of human nucleus pulposus cells forming nucleus pulposus-like tissue in vitro: influence on the proliferation and proteoglycan production of HNPSV-1 cells

Nucleus pulposus (NP) is responsible for maintaining function and structure of the disc. Scaffolds to culture disc cells three-dimensionally are emphasized in recent reports on development of a new method for treating disc degeneration using cell transplantation and tissue engineering. Among artificial scaffolds and cell carrying materials, Atelocollagen is a collagen gel that has an advantage in safety issues over others. However, to date there has been no study that investigated culture of human nucleus pulposus cells in Atelocollagen. To investigate whether Atelocollagen could be used as a culture scaffold and if it has any effect on cell proliferation and proteoglycan (PG) production, as well as to find the optimal commercially available Atelocollagen for NP cell transplantation and tissue engineering, we cultured human NP cell line HNPSV-1, in three different Atelocollagen and compared with alginate. Furthermore, NP-like tissues were generated using these cells and different Atellocollagen solutions. Results showed that both DNA synthesis and content is significantly greater when cultured in Atelocollagen than in alginate. On the other hand, proteoglycan synthesis and accumulation was significantly greater in alginate compared with the 0.3% Atelocollagen scaffolds; with 3% Atelocollagen, however, results were similar. NP-like tissue generated by Atelocollagen showed good water and proteoglycan preservation. The current study demonstrates that the use of Atelocollagen as an in vitro culture scaffold for three-dimensional culture of human NP cell lines is indeed feasible and moreover, Atelocollagen possesses the potential to become a candidate scaffold for cell transplantation or tissue engineering for the treatment of intervertebral disc degeneration.