Scaffold mesh size affects the osteoblastic differentiation of seeded marrow stromal cells cultured in a flow perfusion bioreactor

In this study, we cultured marrow stromal cells on titanium fiber meshes in a flow perfusion bioreactor and examined the effect of altering scaffold mesh size on cell behavior in an effort to develop a bone tissue construct composed of a scaffold, osteogenic cells, and extracellular matrix. Scaffolds of differing mesh size, that is, distance between fibers, were created by altering the diameter of the mesh fibers (20 or 40 microm) while maintaining a constant porosity. These scaffolds had a porosity of 80% and mesh sizes of 65 microm (20-microm fibers) or 119 microm (40-microm fibers). Cell/scaffold constructs were grown in static culture or under flow for up to 16 days and assayed for osteoblastic differentiation. Cellularity was higher at early time points and Ca2+ deposition was higher at later time points for flow constructs over static controls. The 20-microm mesh had reduced cellularity in static culture. Under flow conditions, mass transport limitations are mitigated allowing uniform cell growth throughout the scaffold, and there was no difference in cellularity between mesh types. There was greater alkaline phosphatase (ALP) activity, osteopontin levels, and calcium under flow at 8 days for the 40-microm mesh compared to the 20-microm mesh. However, by day 16, the trend was reversed, suggesting the time course of differentiation was dependent on scaffold mesh size under flow conditions. However, this dependence was not linear with respect to time; larger mesh size was conducive to early osteoblast differentiation while smaller mesh size was conducive to later differentiation and matrix deposition.     

聚乳酸-羟基乙酸共聚物/硅酸钙三维多孔骨组织工程支架的构建与性能

BACKGROUND: Some disadvantages exsist in commonly used poly(lactic-co-glycolic acid) (PLGA) scaffolds, including acidic degradation products, suboptimal mechanical properties, low pore size, poor porosity and pore connectivity rate and uncontrollable shape. OBJECTIVE: To construct a scaffold with three-dimensional (3D) pores by adding calcium silicate to improve the properties of PLGA, and then detect its degradability, mechanical properties and biocompatibility. METHODS: PLGA/calcium silicate porous composite microspheres were prepared by the emulsion-solvent evaporation method, and PLGA 3D porous scaffold was established by 3D-Bioplotter, and then PLGA/calcium silicate composite porous scaffolds were constructed by combining the microspheres with the scaffold using low temperature fusion technology. The compositions, morphology and degradability of the PLGA/calcium silicate porous composite microspheres and PLGA microspheres, as well as the morphology, pore properties and compression strength of the PLGA 3D scaffolds and PLGA/calcium silicate composite porous scaffolds were measured, respectively. Mouse bone marrow mesenchymal stem cells were respectively cultivated in the extracts of PLGA/calcium silicate porous composite microspheres and PLGA microspheres, and then were respectively seeded onto the PLGA 3D scaffolds and PLGA/calcium silicate composite porous scaffolds. Thereafter, the cell proliferation activity was detected at 1, 3 and 5 days. RESULTS AND CONCLUSION: Regular pores on the PLGA microspheres and internal cavities were formed, and the PH values of the degradation products were improved after adding calcium silicate. The fiber diameter, pore, porosity and average pore size of the composite porous scaffolds were all smaller than those of the PLGA scaffolds. The compression strength and elasticity modulus of the composite porous scaffolds were both higher than those of the PLGA scaffolds (P < 0.05). Bone marrow mesenchymal stem cells grew well in above microsphere extracts and scaffolds. These results indicate that PLGA/calcium silicate composite porous scaffolds exhibit good degradability in vitro, mechanical properties and biocompatibility.     

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.     

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

Fiber diameter and texture of electrospun PEOT/PBT scaffolds influence human mesenchymal stem cell proliferation and morphology, and the release of incorporated compounds

Electrospinning (ESP) has lately shown a great potential as a novel scaffold fabrication technique for tissue engineering. Scaffolds are produced by spinning a polymeric solution in fibers through a spinneret connected to a high-voltage electric field. The fibers are then collected on a support, where the scaffold is created. Scaffolds can be of different shapes, depending on the collector geometry, and have high porosity and high surface per volume ratio, since the deposited fibers vary from the microscale to the nanoscale range. Such fibers are quite effective in terms of tissue regeneration, as cells can bridge the scaffold pores and fibers, resulting in a fast and homogeneous tissue growth. Furthermore, fibers can display a nanoporous ultrastructure due to solvent evaporation. The aim of this study was to characterize electrospun scaffolds from poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) copolymers and to unravel the mechanism of pore formation on the fibers. The effect of different fiber diameters and of their surface nanotopology on cell seeding, attachment, and proliferation was studied. Smooth fibers with diameter of 10microm were found to support an optimal cell seeding and attachment within the micrometer range analyzed. Moreover, a nanoporous surface significantly enhanced cell proliferation and cells spreading on the fibers. The fabrication of ESP scaffolds with incorporated dyes with different molecular dimensions is also reported and their release measured. These findings contribute to the field of cell-material interaction and lead to the fabrication of "smart" scaffolds which can direct cells morphology and proliferation, and eventually release biological signals to properly conduct tissue formation.     

Preparation and characterization of collagen-hydroxyapatite composite used for bone tissue engineering scaffold

In this study, highly porous collagen-HA scaffolds were prepared by solid-liquid phase separation method. Microstructure of the composites was characterized by SEM, TEM and XRD. The results show that collagen-HA scaffolds are porous with three-dimension interconnected fiber microstructure, pore sizes are 50-150 microm, and HA particles are dispersed evenly among collagen fiber. Compared with pure collagen, the mechanical property of collagen-HA composite improves significantly. To gain further insight into cell growth throughout 3D scaffolds, the cell proliferation and attachment on the scaffold in vitro was investigated. The collagen-HA composite has good biocompatibility, and adding HA does not affect the histocompatibility of the scaffold materials. The porous collagen-HA composite is suitable as scaffold used for bone tissue engineering.     

Fibrous poly(chitosan-g-DL-lactic acid) scaffolds prepared via electro-wet-spinning

DL-lactic acid was grafted onto chitosan to produce poly(chitosan-g-DL-lactic acid)(PCLA) copolymers. These PCLAs were then spun into submicron and/or nanofibers to fabricate scaffolds using an electro-wet-spinning technique. The diameter of fibers in different scaffolds could vary from about 100 nm to around 3 microm. The scaffolds exhibited various pore sizes ranging from about 1 microm to less than 30 microm and different porosities up to 80%. Two main processing parameters, that is, the concentration of PCLA solutions and the composition proportions of coagulation solutions, were optimized for obtaining desired scaffolds with well-controlled structures. The tensile properties of the scaffolds in both dry and hydrated states were examined. Significantly improved tensile strength and modulus for these fibrous scaffolds in their hydrated state were observed. The data collected from in vitro rabbit-fibroblast/scaffold culture showed that there were no substantial differences in the viability, density and distribution of cultured fibroblasts between PCLA scaffolds and pure chitosan scaffolds.     

Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds

The cellular structure of collagen-glycosaminoglycan (CG) scaffolds used in tissue engineering must be designed to meet a number of constraints with respect to biocompatibility, degradability, pore size, pore structure, and specific surface area. The conventional freeze-drying process for fabricating CG scaffolds creates variable cooling rates throughout the scaffold during freezing, producing a heterogeneous matrix pore structure with a large variation in average pore diameter at different locations throughout the scaffold. In this study, the scaffold synthesis process was modified to produce more homogeneous freezing by controlling of the rate of freezing during fabrication and obtaining more uniform contact between the pan containing the CG suspension and the freezing shelf through the use of smaller, less warped pans. The modified fabrication technique has allowed production of CG scaffolds with a more homogeneous structure characterized by less variation in mean pore size throughout the scaffold (mean: 95.9 microm, CV: 0.128) compared to the original scaffold (mean: 132.4 microm, CV: 0.185). The pores produced using the new technique appear to be more equiaxed, compared with those in scaffolds produced using the original technique.     

Effects of fiber orientation and diameter on the behavior of human dermal fibroblasts on electrospun PMMA scaffolds

We used the electrospinning technique to produce fibrous scaffolds of poly(methyl methacrylate) (PMMA). Using a rotating drum, we aligned the fibers and formed multilayered structures where both the fiber spacing and pore size could be varied. We then plated adult human dermal fibroblasts and studied the effect of fiber diameter and orientation on the cell conformation, integrin receptor expression, proliferation, and migration. We found that a critical diameter minimum diameter existed, D0 = 0.97 microm for cell orientation to occur. For D < D0, no big difference in aspect ratio was observed relative to the control samples on PMMA thin film. Hence, we could fabricate substrate patterned with fibers of different diameters where different cell conformations coexisted on the same scaffold. On the other hand, staining for vinculin proteins in the cells indicated that on large diameter fibers and on flat surfaces, the integrin receptors followed the cell perimeter. On the very small diameter surfaces, the receptors were distributed uniformly along the cell. Cell dynamics studies indicated that the proliferation and migration were also affected by the fiber orientation.     

Tailoring fiber diameter in electrospun poly(epsilon-caprolactone) scaffolds for optimal cellular infiltration in cardiovascular tissue engineering

Despite the attractive features of nanofibrous scaffolds for cell attachment in tissue-engineering (TE) applications, impeded cell ingrowth has been reported in electrospun scaffolds. Previous findings have shown that the scaffold can function as a sieve, keeping cells on the scaffold surface, and that cell migration into the scaffold does not occur in time. Because fiber diameter is directly related to the pore size of an electrospun scaffold, the objective of this study was to systematically evaluate how cell delivery can be optimized by tailoring the fiber diameter of electrospun poly(epsilon-caprolactone) (PCL) scaffolds. Five groups of electrospun PCL scaffolds with increasing average fiber diameters (3.4-12.1 microm) were seeded with human venous myofibroblasts. Cell distribution was analyzed after 3 days of culture. Cell penetration increased proportionally with increasing fiber diameter. Unobstructed delivery of cells was observed exclusively in the scaffold with the largest fiber diameter (12.1 microm). This scaffold was subsequently evaluated in a 4-week TE experiment and compared with a poly(glycolic acid)-poly(4-hydroxybutyrate) scaffold, a standard scaffold used successfully in cardiovascular tissue engineering applications. The PCL constructs showed homogeneous tissue formation and sufficient matrix deposition. In conclusion, fiber diameter is a crucial parameter to allow for homogeneous cell delivery in electrospun scaffolds. The optimal electrospun scaffold geometry, however, is not generic and should be adjusted to cell size.     

Preparation and characteristics of hybrid scaffolds composed of beta-chitin and collagen

Hybrid scaffolds composed of beta-chitin and collagen were prepared by combining salt-leaching and freeze-drying methods. The chitin scaffold used as a framework was easily formed into desired shapes with a uniformly distributed and interconnected pore structure with average pore size of 260-330 microm. The mechanical strength and the rate of biodegradation increased with the porosity, which could be modulated by the salt concentration. In addition, atelocollagen solution was introduced into the macropores of the chitin scaffold to improve cell attachment. Web-like collagen fibers fabricated between pores of chitin were produced by a 0.1 wt% collagen solution, whereas a 0.5 wt% collagen solution only coated the surface of the chitin scaffold. After 3 days of culture, fibroblasts cultured in collagen-coated scaffolds were attached at the place where the collagen was fabricated, whereas cells did not attach and aggregate on the scaffold of chitin alone. After 14 days, the fibroblasts showed a good affinity to and proliferation on all collagen-coated chitins.     

The precision structural regulation of PLLA porous scaffold and its influence on the proliferation and differentiation of MC3T3-E1 cells

A thermal-induced phase separation combined sugar template method was used to fabricate the Poly (L-lactide) acid (PLLA) scaffolds with precisely regulated porous structure. The effect of tuned porous structure of scaffolds on osteoblasts proliferation and differentiation was investigated. The results showed that the pore diameters (200–300, 300–400, 400–500 μm), porosity and interconnectivity of PLLA scaffolds can be accurately controlled indicated by scanning electron microscope. The results of cell experiments showed that the porous structure including the pore size and interconnectivity of scaffolds dramatically influence the cell proliferation and differentiation. The scaffold with pore diameter of 400–500 μm exhibited the highest cell viability and alkaline phosphatase activity among all the scaffolds for the MC3T3-E1 cells. The higher cell proliferation and biocompatibility observed in the 400–500 μm scaffold indicated the high selectivity for MC3T3-E1cells on the pore size of scaffold in tissue engineering. The precise control of the porous structure of scaffold may better guide the cell–matrix interaction in the future research.     

Accurately shaped tooth bud cell-derived mineralized tissue formation on silk scaffolds

Based on the successful use of silk scaffolds in bone tissue engineering, we examined their utility for mineralized dental tissue engineering. Four types of hexafluoroisopropanol (HFIP) silk scaffolds-(250 and 550 microm diameter pores, with or without arginine-glycine-aspartic acid (RGD) peptide) were seeded with cultured 4-day postnatal rat tooth bud cells and grown in the rat omentum for 20 weeks. Analyses of harvested implants revealed the formation of bioengineered mineralized tissue that was most robust in 550 microm pore RGD-containing scaffolds and least robust in 250 microm pore sized scaffolds without RGD. The size and shape of the silk scaffold pores appeared to guide mineralized tissue formation, as revealed using polarized light imaging of collagen fiber alignment along the scaffold surfaces. This study is the first to characterize bioengineered tissues generated from tooth bud cells seeded onto silk scaffolds and indicates that silk scaffolds may be useful in forming mineralized osteodentin of specified sizes and shapes.     

Fabrication and biocompatibility of collagen sponge reinforced with poly(glycolic acid) fiber

This article describes an investigation of collagen sponge mechanically reinforced through the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously dispersed at collagen:PGA weight ratios of 1.5, 0.8, 0.4, and 0.2 was freeze-dried, followed by dehydrothermal cross-linking to obtain collagen sponges incorporating PGA fiber to various extents. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. As expected, PGA fiber incorporation enabled the collagen sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly greater for any collagen sponge incorporating PGA fiber than for collagen sponge. The shrinkage of sponge after cell seeding was suppressed by fiber incorporation. It is possible that shrinkage suppression results in the superior cell attachment of sponge incorporating PGA fiber. After subcutaneous implantation into the backs of mice, the residual volume of collagen sponge incorporating PGA fiber was significant compared with that of collagen sponge and increased with a decrease in the collagen:PGA ratio. The greater number of cells infiltrated and deeper infiltration were observed for collagen sponge incorporating PGA fiber implanted subcutaneously. We conclude that the incorporation of PGA fiber is a simple and promising way to reinforce collagen sponge without impairing biocompatibility.     

Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates

Electrospinning is a promising method to construct fused-fiber biomaterial scaffolds for tissue engineering applications, but the efficacy of this approach depends on how substrate topography affects cell function. Previously, it has been shown that linear, parallel raised features with length scales of 0.5-2 microm direct cell orientation through the phenomenon of contact guidance, and enhance phenotypic markers of osteoblastic differentiation. To determine how the linear, random raised features produced by electrospinning affect proliferation and differentiation of osteoprogenitor cells, poly(lactic acid) and poly(ethylene glycol)-poly(lactic acid) diblock copolymers were electrospun with mean fiber diameters of 0.14-2.1 microm onto rigid supports. MC3T3-E1 osteoprogenitor cells cultured on fiber surfaces in the absence of osteogenic factors exhibited a lower cell density after 7 and 14 days of culture than cells cultured on spin-coated surfaces, but cell density increased with fiber diameter. However, in the presence of osteogenic factors (2 mM beta-glycerophosphate, 0.13 mM L-ascorbate-2-phosphate), cell density after 7 and 14 days of culture on fiber surfaces was comparable to or exceeded spin-coated controls, and alkaline phosphatase activity after 14 days was comparable. Examination of cell morphology revealed that cells grown on fibers had smaller projected areas than those on planar surfaces. However, cells attached to electrospun substrates of 2.1 microm diameter fibers exhibited a higher cell aspect ratio than cells on smooth surfaces. These studies show that topographical factors designed into biomaterial scaffolds can regulate spreading, orientation, and proliferation of osteoblastic cells.     

Engineered cellular response to scaffold architecture in a rabbit trephine defect

Tight control of pore architecture in porous scaffolds for bone repair is critical for a fully elucidated tissue response. Solid freeform fabrication (SFF) enables construction of scaffolds with tightly controlled pore architecture. Four types of porous scaffolds were constructed using SFF and evaluated in an 8-mm rabbit trephine defect at 8 and 16 weeks (n = 6): a lactide/glycolide (50:50) copolymer scaffold with 20% w/w tri-calcium phosphate and random porous architecture (Group 1); another identical design made from poly(desaminotyrosyl-tyrosine ethyl ester carbonate) [poly(DTE carbonate)], a tyrosine-derived pseudo-polyamino acid (Group 2); and two poly(DTE carbonate) scaffolds containing 500 microm pores separated by 500-microm thick walls, one type with solid walls (Group 3), and one type with microporous walls (Group 4). A commercially available coralline scaffold (Interpore) with a 486-microm average pore size and empty defects were used as controls. There was no significant difference in the overall amount of bone ingrowth in any of the devices, as found by radiographic analysis, but patterns of bone formation matched the morphology of the scaffold. These results suggest that controlled scaffold architecture can be superimposed on biomaterial composition to design and construct scaffolds with improved fill time.     

In vitro chondrocyte behavior on porous biodegradable poly (e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.     

Novel apatite fiber scaffolds can promote three-dimensional proliferation of osteoblasts in rodent bone regeneration models

We have successfully synthesized hydroxyapatite fibers via a homogenous precipitation method. Using these hydroxyapatite fibers, we have produced the apatite fiber scaffolds (AFS) with well-controlled pore sizes (porosity above 95%). The AFS is relatively simple to synthesize, and its porosity and pore size are controllable. The usefulness of AFS as a scaffold for bone regeneration was evaluated by (1) seeding and culturing cells in the AFS in vitro, (2) implanting the AFS seeded with cells inside the subcutaneous tissue of mice. The AFS had biocompatibility to support cell adhesion, proliferation, and differentiation. Ectopic bone formation could be formed in the AFS at 12 weeks after implantation into the subcutaneous tissue. Because of its high interpore connection, pore diameters, and porosity, it was believed that AFS was an effective scaffold that provided a three-dimensional cell culture environment. In both in vitro and in vivo environments, the more porous AFS was more advantageous in cell proliferation, cell adhesion, proliferating capacity, robust cell differentiation, ultimately inducing bone ingrowth inside the scaffolds.     

In vitro assessment of cell penetration into porous hydroxyapatite scaffolds with a central aligned channel

There is a clinical need for synthetic scaffolds that promote bone regeneration. A common problem encountered when using scaffolds in tissue engineering is the rapid formation of tissue on the outer edge of the scaffold whilst the tissue in the centre becomes necrotic. To address this, the scaffold design should improve nutrient and cell transfer to the scaffold centre. In this study, hydroxyapatite scaffolds with random, open porosity (average pore size of 282+/-11microm, average interconnecting window size of 72+/-4microm) were manufactured using a modified slip-casting methodology with a single aligned channel inserted into the centre. By varying the aligned channel diameter, a series of scaffolds with channel diameters ranging from 170 to 421microm were produced. These scaffolds were seeded with human osteosarcoma (HOS TE85) cells and cultured for 8 days. Analysis of cell penetration into the aligned channels revealed that cell coverage increased with increasing channel diameter; from 22+/-3% in the 170microm diameter channel to 38+/-6% coverage in the 421microm channel. Cell penetration into the middle section of the 421microm diameter channel (average cell area coverage 121x10(3)+/-32x10(3)microm(2)) was significantly greater than that observed within the 170microm channel (average cell area coverage 26x10(3)+/-6x10(3)microm(2)). In addition, the data presented demonstrates that the minimum channel (or pore) diameter required for cell penetration into such scaffolds is approximately 80microm. These results will direct the development of scaffolds with aligned macroarchitecture for tissue engineering bone.