Human dental pulp progenitor cell behavior on aqueous and hexafluoroisopropanol based silk scaffolds

Silk scaffolds have been successfully used for a variety of tissue engineering applications due to their biocompatibility, diverse physical characteristics, and ability to support cell attachment and proliferation. Our prior characterization of 4-day postnatal rat tooth bud cells grown on hexafluoro-2-propanol (HFIP) silk scaffolds showed that the silk scaffolds not only supported osteodentin formation, but also guided the size and shape of the formed osteodentin. In this study, interactions between human dental pulp cells and HFIP and aqueous based silk scaffolds were studied under both in vitro and in vivo conditions. Silk scaffold porosity and incorporation of RGD and DMP peptides were examined. We found that the degradation of aqueous based silk is much faster than HFIP based silk scaffolds. Also, HFIP based silk scaffolds supported the soft dental pulp formation better than the aqueous based silk scaffolds. No distinct hard tissue regeneration was found in any of the implants, with or without additional cells. We conclude that alternative silk scaffold materials, and hDSC pre-seeding cell treatments or sorting and enrichment methods, need to be considered for successful dental hard tissue regeneration.     

Effect of scaffold design on bone morphology in vitro

Silk fibroin is an important polymer for scaffold designs, forming biocompatible and mechanically robust biomaterials for bone, cartilage, and ligament tissue engineering. In the present work, 3D biomaterial matrices were fabricated from silk fibroin with controlled pore diameter and pore interconnectivity, and utilized to engineer bone starting from human mesenchymal stem cells (hMSC). Osteogenic differentiation of hMSC seeded on these scaffolds resulted in extensive mineralization, alkaline phosphatase activity, and the formation of interconnected trabecular- or cortical-like mineralized networks as a function of the scaffold design utilized; allowing mineralized features of the tissue engineered bone to be dictated by the scaffold features used initially in the cell culture process. This approach to scaffold predictors of tissue structure expands the window of applications for silk fibroin-based biomaterials into the realm of directing the formation of complex tissue architecture. As a result of slow degradation inherent to silk fibroin, scaffolds preserved their initial morphology and provided a stable template during the mineralization phase of stem cells progressing through osteogenic differentiation and new extracellular matrix formation. The slow degradation feature also facilitated transport throughout the 3D scaffolds to foster improved homogeneity of new tissue, avoiding regions with decreased cellular density. The ability to direct bone morphology via scaffold design suggests new options in the use of biodegradable scaffolds to control in vitro engineered bone tissue outcomes.     

Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds

Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112-224 microm) in diameter on one side and large pores (400-500 microm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds' geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (microCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry.     

Synthetic scaffold morphology controls human dermal connective tissue formation

Engineering tissues in bioreactors is often hampered by disproportionate tissue formation at the surface of scaffolds. This hinders nutrient flow and retards cell proliferation and tissue formation inside the scaffold. The objective of this study was to optimize scaffold morphology to prevent this from happening and to determine the optimal scaffold geometric values for connective tissue engineering. After comparing lyophilized crosslinked collagen, compression molded/salt leached PEGT/PBT copolymer and collagen-PEGT/PBT hybrid scaffolds, the PEGT/PBT scaffold was selected for optimization. Geometric parameters were determined using SEM, microcomputed tomography, and flow permeability measurements. Fibroblast were seeded and cultured under dynamic flow conditions for 2 weeks. Cell numbers were determined using CyQuant DNA assay, and tissue distribution was visualized in H&E- and Sirius Red-stained sections. Scaffolds 0.5 and 1.5 mm thick showed bridged connected tissue from top-to-bottom, whereas 4-mm-thick scaffolds only revealed tissue ingrowth until a maximum depth of 0.6-0.8 mm. Rapid prototyped scaffold were used to assess the maximal void space (pore size) that still could be filled with tissue. Tissue bridging between fibers was only found at fiber distances < or =401 +/- 60 microm, whereas filling of void spaces in 3D-deposited scaffolds only occurred at distances < or =273 +/- 55 microm. PEGT/PBT scaffolds having similar optimal porosities, but different average interconnected pore sizes of 142 +/- 50, 160 +/- 56 to 191 +/- 69 microm showed comparable seeding efficiencies at day 1, but after 2 weeks the total cell numbers were significantly higher in the scaffolds with intermediate and high interconnectivity. However, only scaffolds with an intermediate interconnectivity revealed homogenous tissue formation throughout the scaffold with complete filling of all pores. In conclusion, significant amount of connective tissue was formed within 14 days using a dynamic culture process that filled all void spaces of a PEGT/PBT scaffolds with the following geometric parameters: thickness 1.5-1.6 mm, pore size range 90-360 microm, and average interconnecting pore size of 160 +/- 56 microm.     

Development of silk-based scaffolds for tissue engineering of bone from human adipose-derived stem cells

Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and its demonstrated ability to support bone formation both in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASCs), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol (HFIP)), pore sizes (250-500 μm vs. 500-1000 μm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes, with decellularized trabecular bone (DCB) included as a "gold standard". The scaffolds were seeded with hASCs and cultured for 7 weeks in osteogenic medium. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400-600 μm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes, as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2 and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.     

The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds

Highly porous scaffolds of poly(lactide-co-glycolide) (PLGA) were prepared by solution-casting/salt-leaching method. The in vitro degradation behavior of PLGA scaffold was investigated by measuring the change of normalized weight, water absorption, pH, and molecular weight during degradation period. Mesenchymal stem cells (MSCs) were seeded and cultured in three-dimensional PLGA scaffolds to fabricate in vitro tissue engineering bone, which was investigated by cell morphology, cell number and deposition of mineralized matrix. The proliferation of seeded MSCs and their differentiated function were demonstrated by experimental results. To compare the reconstructive functions of different groups, mandibular defect repair of rabbit was made with PLGA/MSCs tissue engineering bone, control PLGA scaffold, and blank group without scaffold. Histopathologic methods were used to estimate the reconstructive functions. The result suggests that it is feasible to regenerate bone tissue in vitro using PLGA foams with pore size ranging from 100-250 microm as scaffolding for the transplantation of MSCs, and the PLGA/MSCs tissue engineering bone can greatly promote cell growth and have better healing functions for mandibular defect repair. The defect can be completely recuperated after 3 months with PLGA/MSCs tissue engineering bone, and the contrastive experiments show that the defects could not be repaired with blank PLGA scaffold. PLGA/MSCs tissue engineering bone has great potential as appropriate replacement for successful repair of bone defect.     

Cell proliferation and migration in silk fibroin 3D scaffolds

Pore architecture in 3D polymeric scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for the seeded cells to organize into a functioning tissue. In this report, we investigated the effects of different freezing temperature regimes on silk fibroin protein 3D scaffold pore microstructure. The fabricated scaffolds using freeze-dry technique were used as a 3D model to monitor cell proliferation and migration. Pores of 200–250 μm diameter were formed by slow cooling at temperatures of ?20 and ?80 °C but were found to be limited in porosity and pore interconnectivity as observed through scanning electron microscopic images. In contrast, highly interconnected pores with 96% porosity were observed when silk solutions were rapidly frozen at ?196 °C. A detailed study was conducted to assess the affect of pore size, porosity and interconnectivity on human dermal fibroblast cell proliferation and migration on these 3D scaffolds using confocal microscopy. The cells were observed to migrate within the scaffold interconnectivities and were found to reach scaffold periphery within 28 days of culture. Confocal images further confirmed normal cell attachment and alignment of actin filaments within the porous scaffold matrix with well-developed nuclei. This study indicates rapid freeze-drying technique as an alternative method to fabricate highly interconnected porous scaffolds for developing functional 3D silk fibroin matrices for potential tissue engineering, biomedical and biotechnological applications.     

Photo-patterning of porous hydrogels for tissue engineering

Since pore size and geometry strongly impact cell behavior and in vivo reaction, the ability to create scaffolds with a wide range of pore geometries that can be tailored to suit a particular cell type addresses a key need in tissue engineering. In this contribution, we describe a novel and simple technique to design porous, degradable poly(2-hydroxyethyl methacrylate) hydrogel scaffolds with well-defined architectures using a unique photolithography process and optimized polymer chemistry. A sphere-template was used to produce a highly uniform, monodisperse porous structure. To create a patterned and porous hydrogel scaffold, a photomask and initiating light were employed. Open, vertical channels ranging in size from 360+/-25 to 730+/-70 microm were patterned into approximately 700 microm thick hydrogels with pore diameters of 62+/-8 or 147+/-15 microm. Collagen type I was immobilized onto the scaffolds to facilitate cell adhesion. To assess the potential of these novel scaffolds for tissue engineering, a skeletal myoblast cell line (C2C12) was seeded onto scaffolds with 147 microm pores and 730 microm diameter channels, and analyzed by histology and digital volumetric imaging. Cell elongation, cell spreading and fibrillar formation were observed on these novel scaffolds. In summary, 3D architectures can be patterned into porous hydrogels in one step to create a wide range of tissue engineering scaffolds that may be tailored for specific applications.     

Initial cell pre-cultivation can maximize ECM mineralization by human mesenchymal stem cells on silk fibroin scaffolds

Fast remineralization of bone defects by means of tissue engineering is one of many targets in orthopedic regeneration. This study investigated the influence of a range of pre-culture durations for human bone marrow derived mesenchymal stem cells (hMSC) before inducing differentiation into osteoblast-like cells. The aim was to find the conditions that lead to maximal extracellular matrix (ECM) mineralization, in terms of both amount and best distribution. Additionally, the influence of silk fibroin scaffold pore size on mineralization was assessed. The formation of mineralized ECM by hMSCs cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified for up to 72 days in culture using non-invasive time-lapse micro-computed tomography (micro-CT). ECM mineralization increased linearly 3 weeks after the beginning of the experiment with addition of differentiation medium. Biochemical end-point assays measured the amount of DNA, calcium deposits, alkaline phosphatase activity and cell metabolic activity to corroborate the hypothesis that an initial pre-culture period of hMSCs on silk fibroin scaffolds can accelerate mineralized ECM formation. According to the micro-CT analysis mineralization on silk fibroin scaffolds with pores of 112-224 μm diameter was most efficient with an initial cell pre-culture period of 9 days, showing 6.87±0.81× higher mineralization values during the whole cultivation period than without an initial cell pre-culture period.     

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.     

Role of poly(lactide-co-glycolide) particle size on gas-foamed scaffolds

Macroporous polymeric scaffolds are frequently used in tissue engineering to allow for cell seeding and host cell invasion of the scaffold following implantation. The process of gas foaming/particulate leaching (GF/PL) is one method to form porous three dimensional scaffolds from particulate poly(lactide-co-glycolide) (PLG). The current study was designed to test the hypothesis that the size of the polymer particles used in this process will control the properties of the scaffolds. Scaffolds were prepared from PLG particles of various sizes (less than 75 microm, 75-106 microm, 106-250 microm and 250-425 microm) and subsequently analyzed. Scaffolds formed from large particles (250-425 microm) displayed significantly decreased compressive moduli, as compared to scaffolds fabricated from smaller particles. In addition, these scaffolds have a pore structure that is less interconnected and contains closed pores. Analysis of tissue in-growth, utilizing a novel computer-aided method, demonstrated that scaffolds formed from smaller particle sizes (less than 106 microm) have significantly more tissue penetration than those formed from larger particle sizes (greater than 106 microm). These results indicate that using small PLG particles (less than 106 microm) leads to high elastic moduli, provides a more interconnected pore structure and promotes greater tissue penetration into the scaffolds in vivo.     

Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering

High strength porous scaffolds and mesenchymal stem cells are required for bone tissue engineering applications. Porous titanium scaffolds (TiS) with a regular array of interconnected pores of 1000 microm in diameter and a porosity of 50% were produced using a rapid prototyping technique. A calcium phosphate (CaP) coating was applied to these titanium (Ti) scaffolds with an electrodeposition method. Raman spectroscopy and energy dispersive X-ray analysis showed that the coating consisted of carbonated hydroxyapatite. Cross-sectioned observations by scanning electron microscopy indicated that the coating evenly covered the entire structure with a thickness of approximately 25 microm. The bonding strength of the coating to the substrate was evaluated to be around 25 MPa. Rat bone marrow cells (RBMC) were seeded and cultured on the Ti scaffolds with or without coating. The Alamar Blue assay provided a low initial cell attachment (40%) and cell numbers were similar on both the uncoated and coated Ti scaffolds after 3 days. The Ti scaffolds were subsequently implanted subcutaneously for 4 weeks in syngenic rats. Histology revealed the presence of a mineralized collagen tissue in contact with the implants, but no bone formation. This study demonstrated that porous Ti scaffolds with high strength and defined geometry may be evenly coated with CaP layers and cultured mesenchymal stem cells for bone tissue engineering.     

Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering

Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(l,d-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3 +/- 58.0 microm) rather than smaller (11.15 +/- 11.08 microm) microspheres, generated pores on the order of 200 microm as compared to 20 microm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications.     

Biodegradable polymer scaffolds with well-defined interconnected spherical pore network

Scaffolding plays pivotal role in tissue engineering. In this work, a novel processing technique has been developed to create three-dimensional biodegradable polymer scaffolds with well-controlled interconnected spherical pores. Paraffin spheres were fabricated with a dispersion method, and were bonded together through a heat treatment to form a three-dimensional assembly in a mold. Biodegradable polymers such as PLLA and PLGA were dissolved in a solvent and cast onto the paraffin sphere assembly. After dissolving the paraffin, a porous polymer scaffold was formed. The fabrication parameters were studied in relation to the pore shape, interpore connectivity, pore wall morphology, and mechanical properties of the polymer scaffolds. The compressive modulus of the scaffolds decreased with increasing porosity. Longer heat treatment time of the paraffin spheres resulted in larger openings between the pores of the scaffolds. Foams of smaller pore size (100-200 microm) resulted in significantly lower compressive modulus than that of larger pore sizes (250-350 or 420-500 microm). The PLLA foams had a skeletal structure consisting of small platelets, whereas PLGA foams had homogeneous skeletal structure. The new processing technique can tailor the polymer scaffolds for a variety of potential tissue engineering applications because of the well-controlled architecture, interpore connectivity, and mechanical properties.     

The effect of composition of calcium phosphate composite scaffolds on the formation of tooth tissue from human dental pulp stem cells

Different approaches towards making 3-dimensional (3-D) bioengineered tooth for future replacement therapy have been developed including scaffold-based tooth regeneration. However, selection of optimal scaffold for future clinical application remains a challenge. In the present study, we tested biocompatibility of four different types of 3-D scaffolds for tooth-tissue regeneration, including a pure poly(lactide-co-glycolide) (PLGA) (70/30, mol/mol) scaffold and three types of calcium phosphate contained composites scaffolds that were 50 wt% of PLGA combined with 50 wt% of hydroxyapatite (HA), tricalcium phosphate (TCP) or calcium carbonate hydroxyapatite (CDHA) respectively. These scaffolds were fabricated by the particle leaching in combination with phase separation technology. Surface modification of these scaffolds was further performed by an ammonia plasma treatment and anchorage of collagen technology. Effect of composition of the composite scaffolds on proliferation of human dental pulp stem cells (DPSCs) was accessed using in vitro MTT assay and in vivo BrdU labeling. Differentiation capability of the DPSCs in the scaffolds was analyzed by measurement of the levels of calcified tissue formation and ALP activity. Our results showed that while the calcium phosphate contained compound is able to support regeneration of tooth tissue effectively, the PLGA/TCP scaffold is more appropriate for the proliferation and differentiation of DPSCs. Furthermore, seeding of dissociated 4-dpn rat tooth bud cells on the PLGA/TCP scaffold generated dentin- and pulp-like tissues. Our results demonstrate that the PLGA/TCP scaffold is superior to the other three scaffolds for tooth-tissue regeneration, especially for dentin formation.     

Tissue-engineered hybrid tooth and bone

Tooth loss accompanied by alveolar bone resorption presents a significant clinical problem. We have investigated the utility of a tissue-engineering approach to provide corrective therapies for tooth-bone loss. Hybrid tooth-bone tissues were bioengineered as follows. Tooth implants were generated from pig third molar tooth bud cells seeded onto polyglycolide (PGA) and polyglycolide-colactide (PLGA) scaffolds, and grown for 4 weeks in the omenta of adult rat hosts. Bone implants were generated from osteoblasts induced from bone marrow progenitor cells obtained from the same pig, seeded onto PLGA fused wafer scaffolds, and grown for 10 days in a rotational oxygen-permeable bioreactor system. The tooth and bone implants were harvested, sutured together, reimplanted, and grown in the omenta for an additional 8 weeks. Histological and immunohistochemical analyses of the excised hybrid tooth-bone constructs revealed the presence of tooth tissues, including primary and reparative dentin and enamel in the tooth portion of hybrid tooth-bone implants, and osteocalcin and bone sialoprotein-positive bone in the bone portion of hybrid tooth-bone constructs. Collagen type III-positive connective tissue resembling periodontal ligament and tooth root structures were present at the interface of bioengineered tooth and bone tissues. These results demonstrate the utility of a hybrid tooth-bone tissue-engineering approach for the eventual clinical treatment of tooth loss accompanied by alveolar bone resorption.     

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

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

Indirect solid free form fabrication of local and global porous,biomimetic and composite 3D polymer-ceramic scaffolds

Precise control over scaffold material, porosity, and internal pore architecture is essential for tissue engineering. By coupling solid free form (SFF) manufacturing with conventional sponge scaffold fabrication procedures, we have developed methods for casting scaffolds that contain designed and controlled locally porous and globally porous internal architectures. These methods are compatible with numerous bioresorbable and non-resorbable polymers, ceramics, and biologic materials. Phase separation, emulsion-solvent diffusion, and porogen leaching were used to create poly(L)lactide (PLA) scaffolds containing both computationally designed global pores (500, 600, or 800 microm wide channels) and solvent fashioned local pores (50-100 microm wide voids or 5-10 microm length plates). Globally porous PLA and polyglycolide/PLA discrete composites were made using melt processing. Biphasic scaffolds with mechanically interdigitated PLA and sintered hydroxyapatite regions were fabricated with 500 and 600 microm wide global pores. PLA scaffolds with complex internal architectures that mimicked human trabecular bone were produced. Our indirect fabrication using casting in SFF molds provided enhanced control over scaffold shape, material, porosity and pore architecture, including size, geometry, orientation, branching, and interconnectivity. These scaffolds that contain concurrent local and global pores, discrete material regions, and biomimetic internal architectures may prove valuable for multi-tissue and structural tissue interface engineering.     

许旺细胞在不同孔径丝素蛋白支架上的生长

背景：细胞在生物支架上的生长行为受到支架表面形貌、润湿性、孔径及孔隙率等多种因素影响。 目的：观察许旺细胞在不同孔径丝素蛋白支架上的生长情况。 方法：制备大孔径50~60 µm、小孔径10~   20 µm两种多孔丝素材料。选用许旺细胞永生化细胞R3 [33-10ras3]为种子细胞,当细胞在培养瓶底形成致密单层时即可消化细胞并进行接种实验,将许旺细胞悬液种于不同形貌的多孔丝素材料表面。复合培养1周后,扫描电镜观察许旺细胞的生长形态及增殖等情况。 结果与结论：不同孔径丝素材料的表面可见许旺细胞生长情况不一。在10~20 µm孔径材料支架上,细胞浓度较低,细胞表现为特异的双极性形态,细胞与细胞之间或平行排列,或首尾相连成细胞链;细胞与细胞之间或平行排列,或首尾相连成细胞链;在50~ 60 µm孔径丝素材料支架上,细胞浓度较高,细胞多为球形,单个分散在多孔支架表面,或呈现成团成串葡萄样聚集在孔的底部,未延展成双极性形态,只有极少量生长在孔与孔之间嵴上的细胞呈双极样。说明多孔丝素蛋白支架的孔径对许旺细胞的黏附、生长有一定的影响,许旺细胞更适合生长在孔径略大于胞体直径的支架材料上。