Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide

Hydrophobic polyhydroxyalkanoate (PHA) scaffolds made of a copolyester of 3-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx) were coated with a fusion protein PHA granule binding protein PhaP fused with RGD peptide (PhaP-RGD). Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on/in the scaffolds for formation of articular cartilages derived from chondrogenic differentiation of hBMSCs for cartilage tissue engineering. PhaP-RGD coating led to more homogeneous spread of cells, better cell adhesion, proliferation and chondrogenic differentiation in the scaffolds compared with those of PhaP coated or uncoated scaffolds immerging in serum minus chondrogenic induction medium. In addition, more extracellular matrices were produced by the differentiated cells over a period of 14 days on/in the PhaP-RGD coated scaffolds evidenced by scanning electron microscopy imaging, enhanced expression of chondrocyte specific genes including SOX-9, aggrecan and type II collagen, suggesting the positive effect of RGD on extracellular matrix production. Furthermore, cartilage-specific extracellular substances sulphated glycosaminoglycans (sGAG) and total collagen content found on/in the PhaP-RGD coated scaffolds were significantly more compared with that produced by the control and PhaP only coated scaffolds. Homogeneously distributed chondrocytes-like cells forming cartilage-like matrices were observed on/in the PhaP-RGD coated scaffolds after 3 weeks. The results suggested that PhaP-RGD coated PHBHHx scaffold promoted chondrogenic differentiation of hBMSCs and could support cartilage tissue engineering.     

Differentiation of a fibrin gel encapsulated chondrogenic cell line

Hyaline cartilage has very limited regenerative capacity following damage. Therefore engineered tissue substitutes have been the focus of much research. Our objective was to develop a fibrin-based scaffold as a cell delivery vehicle and template for hyaline cartilage regeneration, and compare its cellular properties against monolayer and pellet culture for chondrogenic cells. The chondrogenic precursor cell line, RCJ 3.1C5.18 (C5.18), was chosen as a test system for evaluating the effect of various culture conditions, including cell encapsulation, on articular chondrogenic cell differentiation. The C5.18 cells in monolayer showed elevated expression of collagen II, an articular chondrogenic marker, but also markers for fibrocartilage differentiation (collagen I and versican) when cultured with chondrogenic medium as compared to basic maintenance medium. Pellets of C5.18 cells cultured in chondrogenic medium were histologically more organized in structure than pellets cultured in control maintenance medium. The chondrogenic medium cultured pellets also secreted an extracellular matrix that was comprised of type II with very little type I collagen, indicating a trend towards a more hyaline-like cartilage. Moreover, when cultured in chondrogenic medium, fibrin-encapsulated C5.18 cells elaborated an extracellular matrix containing type II collagen, as well as aggrecan, which are both components of hyaline cartilage. This indicated a more articular-like chondrogenic differentiation for fibrin encapsulated C5.18 cells. The results of these experiments provide evidence that the C5.18 cell line can be used as a tool to evaluate potential scaffolds for articular cartilage tissue engineering.     

Chitosan/polyester-based scaffolds for cartilage tissue engineering: Assessment of extracellular matrix formation

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds’ porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold.     

Microscale versus nanoscale scaffold architecture for mesenchymal stem cell chondrogenesis

Nanofiber scaffolds, produced by the electrospinning technique, have gained widespread attention in tissue engineering due to their morphological similarities to the native extracellular matrix. For cartilage repair, studies have examined their feasibility; however these studies have been limited, excluding the influence of other scaffold design features. This study evaluated the effect of scaffold design, specifically examining a range of nano to micron-sized fibers and resulting pore size and mechanical properties, on human mesenchymal stem cells (MSCs) derived from the adult bone marrow during chondrogenesis. MSC differentiation was examined on these scaffolds with an emphasis on temporal gene expression of chondrogenic markers and the pluripotent gene, Sox2, which has yet to be explored for MSCs during chondrogenesis and in combination with tissue engineering scaffolds. Chondrogenic markers of aggrecan, chondroadherin, sox9, and collagen type II were highest for cells on micron-sized fibers (5 and 9?μm) with pore sizes of 27 and 29?μm, respectively, in comparison to cells on nano-sized fibers (300?nm and 600 to 1400?nm) having pore sizes of 2 and 3?μm, respectively. Undifferentiated MSCs expressed high levels of the Sox2 gene but displayed negligible levels on all scaffolds with or without the presence of inductive factors, suggesting that the physical features of the scaffold play an important role in differentiation. Micron-sized fibers with large pore structures and mechanical properties comparable to the cartilage ECM enhanced chondrogenesis, demonstrating architectural features as well as mechanical properties of electrospun fibrous scaffolds enhance differentiation.     

An injectable cross-linked scaffold for nucleus pulposus regeneration

Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering strategies. However with regard to intervertebral disc tissue engineering, the use of a scaffold containing the principal extracellular matrix components of native disc tissue (i.e. collagen type II, aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus pulposus cells that were seeded within non-cross-linked and enzymatically cross-linked, atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any negative effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds retained the highest proteoglycan synthesis rate and the lowest elution of sulfated glycosaminoglycan into the surrounding medium. From confined compression testing and volume reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure for the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the enzymatically cross-linked, composite collagen-hyaluronan scaffold shows the most potential for developing an injectable cell-seeded scaffold for nucleus pulposus treatment in degenerated intervertebral discs.     

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.     

Controlling the porosity of fibrous scaffolds by modulating the fiber diameter and packing density

Porosity has been shown to be a key determinant of the success of tissue engineered scaffolds. A high degree of porosity and an appropriate pore size are necessary to provide adequate space for cell spreading and migration as well as to allow for proper exchange of nutrients and waste between the scaffold and the surrounding environment. Electrospun scaffolds offer an attractive approach for mimicking the natural extracellular matrix (ECM) for tissue engineering applications. The efficacy of electrospinning is likely to depend on the interaction between cells and the geometric features and physicochemical composition of the scaffold. A major problem in electrospinning is the tendency of fibers to accumulate densely, resulting in poor porosity and small pore size. The porosity and pore sizes in the electrospun scaffolds are mainly dependent on the fiber diameter and their packing density. Here we report a method of modulating porosity in three dimensional (3D) scaffolds by simultaneously tuning the fiber diameter and the fiber packing density. Nonwoven poly(ε-caprolactone) mats were formed by electrospinning under various conditions to generate sparse or highly dense micro- and nanofibrous scaffolds and characterized for their physicochemical and biological properties. We found that microfibers with low packing density resulted in improved cell viability, proliferation and infiltration compared to tightly packed scaffolds.     

Assessment of the suitability of chitosan/polybutylene succinate scaffolds seeded with mouse mesenchymal progenitor cells for a cartilage tissue engineering approach

In this work, scaffolds derived from a new biomaterial originated from the combination of a natural material and a synthetic material were tested for assessing their suitability for cartilage tissue engineering applications. In order to obtain a better outcome result in terms of scaffolds' overall properties, different blends of natural and synthetic materials were created. Chitosan and polybutylene succinate (C-PBS) 50/50 (wt%) were melt blended using a twin-screw extruder and processed into 5 x 5 x 5 mm scaffolds by compression moulding with salt leaching. Micro-computed tomography analysis calculated an average of 66.29% porosity and 92.78% interconnectivity degree for the presented scaffolds. The salt particles used ranged in size between 63 and 125 mum, retrieving an average pore size of 251.28 mum. Regarding the mechanical properties, the compressive modulus was of 1.73 +/- 0.4 MPa (E(sec) 1%). Cytotoxicity evaluation revealed that the leachables released by the developed porous structures were not harmful to the cells and hence were noncytotoxic. Direct contact assays were carried out using a mouse bone marrow-derived mesenchymal progenitor cell line (BMC9). Cells were seeded at a density of 5 x 10(5) cells/scaffold and allowed to grow for periods up to 3 weeks under chondrogenic differentiating conditions. Scanning electron microscopy analysis revealed that the cells were able to proliferate and colonize the scaffold structure, and MTS test demonstrated cell viability during the time of the experiment. Finally, Western blot performed for collagen type II, a natural cartilage extracellular matrix component, showed that this protein was being expressed by the end of 3 weeks, which seems to indicate that the BMC9 cells were being differentiated toward the chondrogenic pathway. These results indicate the adequacy of these newly developed C-PBS scaffolds for supporting cell growth and differentiation toward the chondrogenic pathway, suggesting that they should be considered for further studies in the cartilage tissue engineering field.     

Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffolds

Porous scaffold materials that can provide a framework for the cells to adhere, proliferate, and create extracellular matrix are considered to be suitable materials for bone regeneration. Interconnected porous chitosan scaffolds were prepared by freeze-drying method, and were mineralized by calcium and phosphate solution by double-diffusion method to form nanoapatite in chitosan matrix. The mineralized chitosan scaffold contains hydroxyapatite nanocrystals on the surface and also within the pore channels of the scaffold. To assess the effect of apatite and porosity of the scaffolds on cells, human osteoblast (SaOS-2) cells were cultured on unmineralized and mineralized chitosan scaffolds. The cell growth on the mineralized scaffolds and on the pure chitosan scaffold shows a similar growth trend. The total protein content and alkaline phosphatase enzyme activity of the cells grown on scaffolds were quantified, and were found to increase over time in mineralized scaffold after 1 and 3 weeks of culture. The electron microscopy of the cell-seeded scaffolds showed that most of the outer macropores became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed extra cellular matrix, consisting mainly of collagen in mineralized scaffolds. The hydroxyproline content also confirmed the formation of the collagen matrix by cells in mineralized scaffolds. This study demonstrated that the presence of apatite nanocrystals in chitosan scaffolds does not significantly influence the growth of cells, but does induce the formation of extracellular matrix and therefore has the potential to serve for bone tissue engineering.     

An ex vivo model for chondrogenesis and osteogenesis

Loss of bone and cartilage are major healthcare issues. At present, there is a paucity of therapies for effectively repairing these tissues sustainably in the long term. A tissue engineering approach using advanced functional scaffolds may provide a clinically acceptable alternative. In this study, an innovative mineralized alginate/chitosan scaffold was used to provide tailored microenvironments for driving chondrogenesis and osteogenesis from single and mixed populations of human articular chondrocytes and human bone marrow stromal cells. Polysaccharide capsules were prepared with combinations of these cell types with the addition of type I or type II collagen to augment cell-matrix interactions and promote the formation of phenotypically distinct tissues and placed in a rotating (Synthecon) bioreactor or held in static 2D culture conditions for up to 28 days. Significant cell-generated matrix synthesis was observed in human bone marrow bioreactor samples containing type I collagen after 21-28 days, with increased cell proliferation, cell activity and osteocalcin synthesis. The cell-generated matrix was immuno-positive for types I and II collagen, bone sialoprotein and type X collagen, a marker of chondrogenic hypertrophy, demonstrating the formation of a mature chondrogenic phenotype with areas of new osteoid tissue formation. We present a unique approach using alginate/collagen capsules encapsulated in chitosan to promote chondrogenic and osteogenic differentiation and extracellular matrix formation and the potential for tissue-specific differentiation. This has significant implications for skeletal regeneration and application.     

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.     

兔肋软骨脱细胞基质的制备

背景：研究表明新西兰兔软骨组织可作为组织工程支架材料,其中关节软骨及耳软骨的脱细胞基质的研究较多,但采用肋软骨作为组织工程软骨支架的研究较少。 目的：制备新西兰兔肋软骨脱细胞基质,探讨天然软骨支架作为组织工程支架的可行性。 方法：用联合去垢剂-酶法获得软骨支架,根据脱细胞过程中Triton X-100第2次处理时间0,24,48,96 h分为4组。脱细胞完毕后各组支架固定行扫描电镜采集图像观察计算支架孔隙率、孔径长度,并对支架进行苏木精-伊红染色、甲苯胺蓝及Ⅱ型胶原免疫组织化学染色,并将脱细胞支架植入异体新西兰兔皮下观察其相容性。 结果与结论：兔肋软骨脱细胞基质呈乳白色,大小均一,染色示支架结构完整,仍保存大量酸性黏多糖及Ⅱ型胶原成分,扫描电镜观察经一定时间的脱细胞处理后可得到结构完整,孔隙均匀的天然软骨支架,其孔隙率为(61.31±8.45) %;孔径长度为(32.80±5.15) μm,符合正态性分布,各组脱细胞支架植入异体新西兰兔皮下7 d后生物相容性良好,周围软组织无明显充血、化脓等炎症排斥反应出现。结果显示,兔肋软骨脱细胞支架具有良好的基质组成,有较完整、均匀的孔隙结构及孔径分布,可作为组织工程支架材料。     

Nano-fibrous poly(L-lactic acid) scaffolds with interconnected spherical macropores

Biodegradable polymers have been used extensively as scaffolding materials to regenerate new tissues. These scaffolds should possess certain physical characteristics including a three-dimensional structure, high porosity with an interconnected pore structure, and a suitable surface structure for cell attachment, proliferation, and differentiation. To mimic the fibrous architecture of type I collagen, nano-fibrous matrices have been created in our laboratory using a phase-separation technique of poly(L-lactic acid) (PLLA) solutions. In addition, biodegradable scaffolds with controlled interconnected spherical pore networks have been fabricated in our laboratory. In this work, these two techniques were combined to yield scaffolds with highly interconnected spherical macroporous structures and nano-fibrous architectures. Paraffin spheres were first fabricated with a dispersion method, and were thermally bonded to form an interconnected mold. PLLA solutions were cast over the paraffin sphere assembly and were thermally phase-separated to form nano-fibrous matrices. After leaching out the paraffin, synthetic nano-fibrous extracellular matrices with interconnected spherical pores were yielded. By utilizing this fabrication process, we are able to control the architecture of the scaffolds at several different levels, including the macroscopic shape of the scaffold, the spherical pore size, interfiber distance, and the fiber diameter at the nano-size scale. The inter-pore connectivity could be controlled by varying the heat treatment time of the paraffin spheres, and mechanical properties could be controlled by varying the porosity of the scaffolds. With an interconnected macroporous structure that promotes cell seeding throughout the interstices of the scaffold, and a synthetic collagen-like matrix, these novel matrices may be an excellent scaffold for tissue engineering.     

Three-dimensional duck’s feet collagen/PLGA scaffold for chondrification: role of pore size and porosity

Abstract

An ideal tissue-engineered scaffold must provide sufficient porosity to allow free movement of cells, nutrients, and oxygen for proper cell growth and further maintenance. Owing to variation in pore sizes and shapes of as-fabricated scaffold, the amount of oxygen available for the cells attached to the scaffold and transfer of by-products and excrement will be different, which ultimately results in cell activity. Thus, optimizing pore size and porosity of a scaffold for a specific tissue regeneration are one of the key highlights, which should be considered while designing a scaffold as well as choosing a specific cell type. In this study, three-dimensional (3D) scaffolds based on blends of duck’s feet collagen (DC) and poly (lactic-co-glycolic acid) (PLGA) with different pore sizes i.e. 90–180, 180–250, 250–355 and 355–425 μm were prepared using solvent casting/salt leaching approach and examined its effects on chondrification. The morphological analysis of the as-fabricated scaffolds was performed using SEM for studying porosity and pore size. The cell proliferation and gene expression were investigated after culturing costal chondrocytes on each scaffolds using 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and qRT-PCR. Histological staining of in vivo implants was performed in nude mice as models. The biological evaluation showed a pore-size dependent chondrification at different time points. Especially, the 355–425 μm DC/PLGA scaffold showed a highest positive impact on maintenance of cell proliferation, costal chondrocyte phenotype and increased glycosaminoglycan accumulation than the other groups. These results indicated that DC/PLGA scaffolds with pore size ranging from 250 to 425 μm can be considered as highly-suitable constructs for enhanced chondrification.     

TGF-beta1 immobilized tri-co-polymer for articular cartilage tissue engineering

Tri-co-polymer with composition of gelatin, hyaluronic acid and chondroitin-6-sulfate has been used to mimic the cartilage extracellular matrix as scaffold for cartilage tissue engineering. In this study, we try to immobilize TGF-beta1 onto the surface of the tri-co-polymer sponge to suppress the undesired differentiation during the cartilage growth in vitro. The scaffold was synthesized with a pore size in a range of 300-500 microm. TGF-beta1 was immobilized on the surface of the tri-co-polymer scaffold with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Tri-co-polymer scaffolds with and without TGF-beta1 were seeded with porcine chondrocytes and cultured in a spinner flask for 2, 4, and 6 weeks. The chondrocytes were characterized by the methods of immunohistochemical staining with anti-type II collagen and anti-S-100 protein monoclonal antibody, and RT-PCR. After culturing for 4 weeks, chondrocytes showed positive in S-100 protein, Alcian blue, and type II collagen for the scaffold with TGF-beta1 immobilization. There is no observed type I and type X collagen expression in the scaffolds from the observation of RT-PCR. In addition, the scaffold without TGF-beta1 immobilization, type X collagen, can be detected after cultured for 2 weeks. Type I collagen was progressively expressed after 4 weeks. These results can conclude that TGF-beta1 immobilized scaffold can suppress chondrocytes toward prehypertrophic chondrocytes and osteolineage cells. The tri-co-polymer sponge with TGF-beta1 immobilization should have a great potential in cartilage tissue engineering in the future.     

Application of an elastic biodegradable poly(L-lactide-co-epsilon-caprolactone) scaffold for cartilage tissue regeneration

In cartilage tissue regeneration, it is important that an implant inserted into a defect site can maintain its mechanical integrity and endure stress loads from the body, in addition to being biocompatible and able to induce tissue growth. These factors are crucial in the design of scaffolds for cartilage tissue engineering. We developed an elastic biodegradable scaffold from poly(L-lactideco-epsilon-caprolactone) (PLCL) for application in cartilage treatment. Biodegradable PLCL co-polymer was synthesized from L-lactide and epsilon-caprolactone in the presence of stannous octoate as a catalyst. A highly elastic PLCL scaffold was fabricated by a gel-pressing method with 80% porosity and 300-500 microm pore size. The tensile mechanical and recovery tests were performed in order to examine mechanical and elastic properties of the PLCL scaffold. They could be easily twisted and bent and exhibited almost complete (over 94%) recoverable extension up to breaking point. For examining cartilaginous tissue formation, rabbit chondrocytes were seeded on scaffolds. They were then cultured in vitro for 5 weeks or implanted in nude mice subcutaneously. From in vitro and in vivo tests, the accumulation of extracellular matrix on the constructs showed that chondrogenic differentiation was sustained onto PLCL scaffolds. Histological analysis showed that cells onto PLCL scaffolds formed mature and well-developed cartilaginous tissue, as evidenced by chondrocytes within lacunae. From these results, we are confident that elastic PLCL scaffolds exhibit biocompatibility and as such would provide an environment where cartilage tissue growth is enhanced and facilitated.     

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.     

Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration

Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.     

Polymeric Scaffolds for Bone Tissue Engineering

Bone tissue engineering is a rapidly developing area. Engineering bone typically uses an artificial extracellular matrix (scaffold), osteoblasts or cells that can become osteoblasts, and regulating factors that promote cell attachment, differentiation, and mineralized bone formation. Among them, highly porous scaffolds play a critical role in cell seeding, proliferation, and new 3D-tissue formation. A variety of biodegradable polymer materials and scaffolding fabrication techniques for bone tissue engineering have been investigated over the past decade. This article reviews the polymer materials, scaffold design, and fabrication methods for bone tissue engineering. Advantages and limitations of these materials and methods are analyzed. Various architectural parameters of scaffolds important for bone tissue engineering (e.g. porosity, pore size, interconnectivity, and pore-wall microstructures) are discussed. Surface modification of scaffolds is also discussed based on the significant effect of surface chemistry on cells adhesion and function.