A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells

We developed a natural, acellular, 3-D interconnected porous scaffold derived from cartilage extracellular matrix (ECM). Human cartilage was physically shattered, then decellularized sequentially with use of hypotonic buffer, TritonX-100, and a nuclease solution and made into a suspension. The scaffold was fabricated by simple freeze-drying and cross-linking techniques. On histology, scaffolds showed most of the ECM components after removal of the cell fragments, and scanning electron microscopy revealed a 3-D interconnected porous structure. Cellular viability assay revealed no cytotoxic effects. In vitro study showed that the novel scaffold could provide a suitable 3-D environment to support the adheration, proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to chondrocytes in culture with chondrogenic medium after 21 days. Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds and implanted subcutaneously into nude mice. Four weeks later, cartilage-like tissue formed, with positive staining for Safranin O, tuoluidine blue and collagen II. Cells in the samples seemed to confirm that they originated from the labeled BMSCs, as confirmed by in vivo fluorescent imaging and immunofluorescence examination. In conclusion, the cartilage ECM-derived porous scaffold shows potential as biomaterial for cartilage tissue engineering, and PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo.     

Cartilage regeneration with highly-elastic three-dimensional scaffolds prepared from biodegradable poly(L-lactide-co-epsilon-caprolactone)

Compressive mechanical stimuli are crucial in regenerating cartilage with tissue engineering, which creates a need for scaffolds that can maintain their mechanical integrity while delivering mechanical signals to adherent cells during strain applications. With these goals in mind, the aim of this study was to develop a mechano-active scaffold that facilitated effective cartilaginous tissue formation under dynamic physiological environments. Using a gel-pressing method, we fabricated a biodegradable and highly-elastic scaffold from poly(L-lactide-co-epsilon-caprolactone) (PLCL; 5:5), with 85% porosity and a 300-500-microm pore size, and we compared it to control scaffolds made of rigid polylactide (PLA) or poly(lactide-co-glycolide) (PLGA). After tensile mechanical tests and recovery tests confirmed the elasticity of the PLCL scaffolds, we seeded them with rabbit chondrocytes, cultured them in vitro, and subcutaneously implanted them into nude mice for up to eight weeks. The PLCL scaffolds possessed a completely rubber-like elasticity, were easily twisted and bent, and exhibited an almost complete (over 97%) recovery from applied strain (up to 500%); the control PLA scaffolds showed little recovery. In vitro and in vivo accumulations of extracellular matrix on the cell-PLCL constructs demonstrated that they could not only sustain but also significantly enhance chondrogenic differentiation. Moreover, the mechanical stimulation of the dynamic in vivo environment promoted deposition of the chondral extracellular matrix onto the PLCL. In contrast, on the PLA scaffolds, most of the chondrocytes had de-differentiated and formed fibrous tissues. In a rabbit defect model, the groups treated with PLCL scaffolds exhibited significantly enhanced cartilage regeneration compared to groups harboring an empty control or PLGA scaffolds. These results indicated that the mechano-active PLCL scaffolds effectively delivered mechanical signals associated with biological environments to adherent chondrocytes, suggesting that these elastic PLCL scaffolds could successfully be used for cartilage regeneration.     

Engineering ear constructs with a composite scaffold to maintain dimensions

Engineered cartilage composed of a patient's own cells can become a feasible option for auricular reconstruction. However, distortion and shrinkage of ear-shaped constructs during scaffold degradation and neocartilage maturation in vivo have hindered the field. Scaffolds made of synthetic polymers often generate degradation products that cause an inflammatory reaction and negatively affect neocartilage formation in vivo. Porous collagen, a natural material, is a promising candidate; however, it cannot withstand the contractile forces exerted by skin and surrounding tissue during normal wound healing. We hypothesised that a permanent support in the form of a coiled wire embedded into a porous collagen scaffold will maintain the construct's size and ear-specific shape. Half-sized human adult ear-shaped fibrous collagen scaffolds with and without embedded coiled titanium wire were seeded with sheep auricular chondrocytes, cultured in vitro for up to 2 weeks, and implanted subcutaneously on the backs of nude mice. After 6 weeks, the dimensional changes in all implants with wire support were minimal (2.0% in length and 4.1% in width), whereas significant reduction in size occurred in the constructs without embedded wire (14.4% in length and 16.5% in width). No gross distortion occurred over the in vivo study period. There were no adverse effects on neocartilage formation from the embedded wire. Histologically, mature neocartilage extracellular matrix was observed throughout all implants. The amount of DNA, glycosaminoglycan, and hydroxyproline in the engineered cartilage were similar to that of native sheep ear cartilage. The embedded wire support was essential for avoiding shrinkage of the ear-shaped porous collagen constructs.     

Three-dimensional perfusion culture of human adipose tissue-derived endothelial and osteoblastic progenitors generates osteogenic constructs with intrinsic vascularization capacity

In this study, we aimed at generating osteogenic and vasculogenic constructs starting from the stromal vascular fraction (SVF) of human adipose tissue as a single cell source. SVF cells from human lipoaspirates were seeded and cultured for 5 days in porous hydroxyapatite scaffolds by alternate perfusion through the scaffold pores, eliminating standard monolayer (two-dimensional [2D]) culture. The resulting cell-scaffold constructs were either enzymatically treated to extract and characterize the cells or subcutaneously implanted in nude mice for 8 weeks to assess the capacity to form bone tissue and blood vessels. SVF cells were also expanded in 2D culture for 5 days and statically loaded in the scaffolds. The SVF yielded 5.9 +/- 3.5 x 10(5) cells per milliliter of lipoaspirate containing both mesenchymal progenitors (5.2% +/- 0.9% fibroblastic colony forming units) and endothelial-lineage cells (54% +/- 6% CD34+/CD31+ cells). After 5 days, the total cell number was 1.8-fold higher in 2D than in three-dimensional (3D) cultures, but the percentage of mesenchymal- and endothelial-lineage cells was similar (i.e., 65%-72% of CD90+ cells and 7%-9% of CD34+/CD31+ cells). After implantation, constructs from both conditions contained blood vessels stained for human CD31 and CD34, functionally connected to the host vasculature. Importantly, constructs generated under 3D perfusion, and not those based on 2D-expanded cells, reproducibly formed bone tissue. In conclusion, direct perfusion of human adipose-derived cells through ceramic scaffolds establishes a 3D culture system for osteoprogenitor and endothelial cells and generates osteogenic-vasculogenic constructs. It remains to be tested whether the presence of endothelial cells accelerates construct vascularization and could thereby enhance implanted cell survival in larger size implants. Disclosure of potential conflicts of interest is found at the end of this article.     

Cartilaginous tissue formation using a mechano-active scaffold and dynamic compressive stimulation

It is known that complex loading is involved in the development and maintenance of articular cartilage in the body. It means the compressive mechanical stimulation is a very important factor for formation of articular cartilage using a tissue-engineering technique. The objective of this study is to engineer cartilaginous constructs with mechano-active scaffolds and to evaluate the effect of dynamic compression for regeneration of cartilage. The mechano-active scaffolds were prepared from a very elastic poly(L-lactide-co-epsilon-caprolactone) (PLCL) with 85% porosity and 300-500 mum pore size using a gel-pressing method. The scaffold was seeded with 2 x 10(6) chondrocytes and the continuous compressive deformation of 5% strain was applied with 0.1 Hz for 10 days and 24 days, respectively. Then, the chondrocytes-seeded constructs were implanted subcutaneously into nude mice. Mechano-active scaffolds with complete rubber-like elasticity showed almost complete (over 97%) recovery at an applied strain of up to 500%. The amount of chondral extracellular matrix was increased significantly by mechanical stimulation on the highly elastic mechano-active scaffolds. Histological analysis showed the mechanically stimulated implants formed mature and well-developed cartilaginous tissue, as evidenced by the chondrocytes within lacunae and the abundant accumulation of sulfated GAGs. However, unhealthy lacunae shapes and hypertrophy forms were observed in the implants stimulated mechanically for 24 days, compared with those stimulated for 10 days. In conclusion, the proper periodical application of dynamic compression can encourage chondrocytes to maintain their phenotypes and enhance the production of GAGs, which would improve the quality of cartilaginous tissue formed both in vitro and in vivo.     

Cartilaginous tissue formation using a mechano-active scaffold and dynamic compressive stimulation

It is known that complex loading is involved in the development and maintenance of articular cartilage in the body. It means the compressive mechanical stimulation is a very important factor for formation of articular cartilage using a tissue-engineering technique. The objective of this study is to engineer cartilaginous constructs with mechano-active scaffolds and to evaluate the effect of dynamic compression for regeneration of cartilage. The mechano-active scaffolds were prepared from a very elastic poly(L-lactide-co-ε-caprolactone) (PLCL) with 85% porosity and 300–500 μm pore size using a gel-pressing method. The scaffold was seeded with 2 × 10⁶ chondrocytes and the continuous compressive deformation of 5% strain was applied with 0.1 Hz for 10 days and 24 days, respectively. Then, the chondrocytes-seeded constructs were implanted subcutaneously into nude mice. Mechano-active scaffolds with complete rubber-like elasticity showed almost complete (over 97%) recovery at an applied strain of up to 500%. The amount of chondral extracellular matrix was increased significantly by mechanical stimulation on the highly elastic mechano-active scaffolds. Histological analysis showed the mechanically stimulated implants formed mature and well-developed cartilaginous tissue, as evidenced by the chondrocytes within lacunae and the abundant accumulation of sulfated GAGs. However, unhealthy lacunae shapes and hypertrophy forms were observed in the implants stimulated mechanically for 24 days, compared with those stimulated for 10 days. In conclusion, the proper periodical application of dynamic compression can encourage chondrocytes to maintain their phenotypes and enhance the production of GAGs, which would improve the quality of cartilaginous tissue formed both in vitro and in vivo.     

聚乳酸-聚乙醇酸可降解支架构建组织工程食管

目的:观察人食管上皮细胞在聚乳酸-聚乙醇酸(PLGA)支架上的贴附和生长情况,利用组织工程技术培养工程化人工食管.方法:制作PLGA三维细胞支架;分离培养成人食管上皮细胞,体外扩增后种植到PLGA支架上.在体外和裸鼠体内分别培养食管上皮细胞-支架复合物,分期终止培养,进行组织学染色、扫描电镜、细胞角蛋白免疫组织化学检测.结果:体外培养显示,人食管上皮细胞在支架材料上贴附生长良好,长期培养仍保持食管上皮细胞特性,裸鼠体内培养4周后可形成食管黏膜样组织.结论:PLGA支架适合食管上皮细胞黏附生长,可作为食管组织工程的细胞载体.     

Fabrication and characterization of hydrophilic poly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-molding particulate-leaching method

Porous PLGA/PVA scaffolds were fabricated by blending poly(lactic-co-glycolic acid) (PLGA) with polyvinyl alcohol (PVA) to improve the hydrophilicity and cell compatibility of the scaffolds for tissue engineering applications. PLGA/PVA blend scaffolds with different PVA compositions up to 20wt% were fabricated by a melt-molding particulate-leaching method (non-solvent method). The prepared scaffolds were investigated by scanning electron microscopy (SEM), mercury intrusion porosimetry, the measurements of water contact angles and bi-axial tensile strengths, etc. for their surface and bulk characterizations. The scaffolds exhibited highly porous and open-cellular pore structures with almost same surface and interior porosities (pore size, 200-300 microm; porosity, about 90%). The PLGA/PVA blend scaffolds with PVA compositions more than 5% were easily wetted in cell culture medium without any prewetting treatments, which is highly desirable for tissue engineering applications. In vitro cell compatibility of the control hydrophobic PLGA and hydrophilized PLGA/PVA (5wt%) blend scaffolds was compared by the culture of human chondrocytes in the scaffolds and the following analyses by MTT assay and SEM observation. It was observed that the PLGA/PVA blend scaffold had better cell adhesion and growth than the control PLGA scaffold. For in vivo evaluation of tissue compatibility, the scaffolds were implanted into the skull defects of rabbits. The results were evaluated by histology examinations. The PLGA/PVA (5wt%) blend scaffold showed better bone ingrowth into the scaffold and new bone formation inside the scaffold than the PLGA scaffold. It seems that 5% addition of PVA to PLGA to fabricate PLGA/PVA blend scaffolds is enough for improving the hydrophilicity and cell compatibility of the scaffolds.     

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.     

Effects of cross-linking type II collagen-GAG scaffolds on chondrogenesis in vitro: dynamic pore reduction promotes cartilage formation

Articular cartilage tissue-engineering investigations often implement bioassays for chondrogenesis in vitro using articular chondrocytes or mesenchymal stem cells in cell pellets that contract with time in culture, suggesting an association between the processes of contraction of the cell pellet and cartilage formation. The objective of the present study was to investigate this relationship further using adult canine articular chondrocyte-seeded type II collagen-GAG scaffolds. The collagen-GAG scaffolds were chemically cross-linked to achieve a range of cross-link densities. Chondrocyte-seeded scaffolds of varying cross-link densities were then cultured for 2 weeks to evaluate the effect of crosslink density on scaffold contraction and chondrogenesis. Scaffolds with low cross-link densities experienced cell-mediated contraction, increased cell number densities, a greater degree of chondrogenesis (viz., chondrocytic morphology of cells, synthesis of type II collagen), and an apparent increase in the rate of degradation of the scaffold compared to more highly cross-linked scaffolds that resisted cellular contraction. The results of this study suggest the promise of "dynamic pore reduction" for scaffolds for articular cartilage tissue engineering. In this approach, scaffolds would have an initial pore diameter large enough to facilitate cell seeding and a mechanical stiffness low enough to allow for cell-mediated contraction to yield a reduced pore volume to favor chondrogenesis. This approach may provide a useful alternative to traditional means of increasing cell number density and retention of synthesized molecules that promote cartilage formation in tissue-engineered constructs.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Enhancement of osteogenesis by poly(lactide-co-glycolide) sponges loaded with surface-embedded hydroxyapatite particles and rhBMP-2

The bone mesenchymal stem cells (BMSCs) were seeded on [poly(lactide-co-glycolide) scaffolds with hydroxyapatite (HA) coating, and "s" stands for surface] (PLGA/HA-S), PLGA/HA-M (containing the same HA amount in the matrix as that of the PLGA/HA-S and "m" stands for matrix), and PLGA scaffolds, which were then cultured in a medium-containing Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2). In vitro culture of rat BMSCs found no different cell morphology in all the scaffolds, but the alkaline phosphatase activity and osteogenic gene expression of type I collagen (COL I) and osteocalcin (OCN) in the PLGA/HA-S scaffolds were always highest and were significantly improved in comparison with those in the PLGA scaffolds. In a rat calvarial defect model, new bone formation was enhanced in the PLGA/HA-S/ErhBMP-2 implants at 4 and 8 weeks after implantation too. Therefore, the PLGA/HA-S scaffold can better enhance the ErhBMP-2-induced osteogenic differentiation of BMSCs in vitro and osteogenesis in vivo.     

Chondroconductive potential of tantalum trabecular metal

Mesenchymal stem cells or chondrocytes have been implanted into joints in biodegradable matrices in order to improve the quality of healing cartilage defects; however, insufficient biomechanical strength of the construct at implantation is a limiting factor for clinical application. Logically, a construct with better biomechanical characteristics would provide better results. Tantalum trabecular metal (TTM) is osteoconductive and mechanically similar to subchondral bone. The objective of this pilot study was to determine if TTM is also chondroconductive. Small sections of TTM were cultured with emu and canine chondrocytes in static and dynamic culture environments. The sections cultured in dynamic bioreactors were diffusely covered with a cartilaginous matrix. Sections cultured in static conditions had no growth. Histologic evaluation from emu and canine dynamic cultures showed tissue that was heavily populated with mesenchymal cells that resembled chondrocytes, and glycosaminoglycan staining that was distributed throughout the matrix. Type II collagen content in the canine dynamic culture was 84% by SDS-PAGE. Tantalum trabecular metal is chondroconductive in vitro in a dynamic environment when cultured with adult canine or emu chondrocytes. This technology could be expanded to determine if cartilaginous-metallic constructs may be used for joint resurfacing of osteoarthritic joints.     

Tissue engineering for total meniscal substitution: animal study in sheep model-results at 12 months

The aim of the study was to investigate the use of a hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Eighteen skeletally mature sheep were treated. The animals were divided into three groups: cell-free scaffold, scaffold seeded with autologous chondrocytes, and meniscectomy alone. The implant was sutured to the capsule and to the meniscal ligament. At a 12-month gross assessment, histology and histomorphometry were used to assess the meniscus implant, knee joint, and osteoarthritis development. All implants showed excellent capsular ingrowth at the periphery. The implant gross assessment showed significant differences between cell-seeded and cell-free groups (p=0.011). The histological analysis indicated a cellular colonization throughout the implanted constructs. Avascular cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Joint gross assessment showed that sheep treated with scaffold implantation achieved a significant higher score than those underwent meniscectomy (p<0.0005), and the Osteoarthritis Research Society International score showed that osteoarthritic changes were significantly less in the cell-seeded group than in the meniscectomy group (p=0.047), even though results were not significantly superior to those of the cell-free scaffold. Seeding of the scaffold with autologous chondrocytes increases its tissue regeneration capacity, providing a better fibrocartilaginous tissue formation. The study suggests the potential of the novel hyaluronic acid/polycaprolactone scaffold for total meniscal substitution, although this approach has to be further improved before being applied into clinical practice.     

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.     

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈86% of the defect area and ≈61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.     

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.     

Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds

The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions.     

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.