Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: A model for cell-mechanics studies

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering.     

Positive changes in bone marrow-derived cells in response to culture on an aligned bioscaffold

Previous studies have shown that cultured cells align with the local topography of their substrate following a concept called "contact guidance." Additionally, if the topography is highly aligned, the cells produce newly synthesized matrix that is also aligned. The objective of this study was to elucidate the positive effect of cell seeding on an elongated porcine small intestinal submucosa (SIS), which has been shown to improve ligament and tendon healing, by measuring the cellular response as a result of the changes in alignment. Because elongation is known to align the fibers of SIS through recruitment along the direction of elongation, we hypothesized that rabbit bone marrow-derived cells (BMDCs) seeded on SIS with improved fiber alignment would increase the expression and production of collagen following the concept of contact guidance. Using the small-angle light-scattering technique, it was found that a 15% elongation together with BMDC seeding on SIS (elongated, seeded group) improved its alignment of collagen fibers up to 16 times more than no elongation and no BMDC seeding (non-elongated, non-seeded group). Furthermore, BMDCs were also aligned along the direction of elongation and showed 200% greater collagen type I gene expression in the elongated, seeded group than in Petri dish controls. More importantly, the production of collagen was also 24% greater. The results of this study demonstrate that alignment of a bioscaffold can result in positive changes in cellular response, making the bioscaffold more attractive for functional tissue engineering to potentially enhance healing of ligaments and tendons.     

具有周向微槽结构管状血管支架制备及诱导平滑肌细胞的取向生长

背景：对小口径血管组织工程化而言,平滑肌细胞的周向排列要求彻底改变以前支架的简单多孔结构,代之以能够诱导血管平滑肌细胞三维周向取向和排列新型微观结构。 目的：观察微槽结构对平滑肌细胞体外定向诱导的影响。 方法：用静电纺丝、熔融纺丝并利用溶剂/非溶剂和热压的方式制得了具有两层管壁、外壁具有周向微沟槽结构的仿生管状血管支架,用胶原蛋白固定改性后,在其上种植人脐静脉血管平滑肌细胞。扫描电镜和荧光显微镜观察支架不同缠绕角度对平滑肌细胞定向诱导能力的影响。 结果与结论：①选择比例为5∶95的氯仿/乙醇溶液,浸润时间为5 s,可以使乳酸-羟基乙酸共聚物电纺纤维和乳 酸-ε-己内酯共聚物熔纺纤维之间形成很好的粘连,形成支架。②通过碱降解使支架表面含有羧基,以1-(二甲基胺丙基)-3-乙基碳化二亚胺为缩合剂在支架表面接枝胶原。X射线光电子能谱证实了支架表面胶原大分子的存在。③当纤维之间的编织角度为30°即网孔尺寸适当时,细胞能在支架内部和表面大面积生长。④具有两层管壁结构的仿生管状血管支架具有良好的细胞相容性,其表面周向微槽结构对平滑肌细胞的取向排列具有明显的诱导作用。提示在电纺层外面再熔纺缠绕降解聚合物是制备管状仿生血管支架的可行方法。血管平滑肌细胞能沿着纤维暨微沟槽方向一致取向排列。     

Anisotropic cell sheets for constructing three-dimensional tissue with well-organized cell orientation

Normal human dermal fibroblasts were aligned on micropatterned thermoresponsive surfaces simply by one-pot cell seeding. After they proliferated with maintaining their orientation, anisotropic cell sheets were harvested by reducing temperature to 20 °C. Surprisingly, the cell sheets showed different shrinking rates between vertical and parallel sides of the cell alignment (aspect ratio: approx. 3: 1), because actin fibers in the cell sheets were oriented with the same direction. The control of cell alignment provided not only a physical anisotropy but also biological impacts to the cell sheet. Vascular endothelial growth factor (VEGF) secreted by aligned fibroblasts was increased significantly, whereas transforming growth factor-β1 (TGF-β1) expression was the same level in anisotropic cell sheets as cell sheets having random cell orientations. Furthermore, although the amount of deposited type Ⅰ collagen was different non-significantly onto between cell sheets with and without controlled cell alignment, collagen deposited onto fibroblasts sheets with cell alignment also showed anisotropy, verified by a fluorescence imaging analysis. The physical and biological anisotropies of cell sheets were potentially useful to construct biomimetic tissues that were organized by aligned cells and/or extracellular matrix (ECM) including collagen in cell sheet-based regenerative medicine. Furthermore, due to the unique thermoresponsive property, the anisotropic cell sheets were successfully manipulated using a gelatin-coated plunger and were layered with maintaining their cell alignment. The combined use of the anisotropic cell sheet and cell sheet manipulation technique promises to create complex tissue that requires the three-dimensional control of their anisotropies, as one of the next-generation cell sheet technologies.     

Platelet-derived growth-factor-releasing aligned collagen–nanoparticle fibers promote the proliferation and tenogenic differentiation of adipose-derived stem cells

In order to enhance the healing potential of an injured tendon, we have prepared a novel biomimetic aligned collagen–nanoparticle (NP) composite fiber using an electrochemical process. The aligned collagen–NP composite fiber is designed to affect the cellular activity of adipose-derived stem cells (ADSCs) through two different ways: (i) topographic cues from the alignment of collagen fibril and (ii) controlled release of platelet-derived growth factors (PDGFs) from the NPs. PDGF released from collagen–NP fibers significantly enhanced the proliferation of ADSCs when tested for up to 7 days. Moreover, compared to random collagen fibers with PDGFs, aligned collagen–NP fibers significantly promoted the desirable tenogenic differentiation of ADSCs, as evidenced by an increased level of tendon markers such as tenomodulin and scleraxis. On the other hand, no undesirable osteogenic differentiation, as measured by the unchanged level of alkaline phosphatase and osteocalcin, was observed. Together, these results indicate that the aligned collagen–NP composite fiber induced the tenogenic differentiation of ADSCs through both a topographic cue (aligned collagen fibril) and a chemical cue (PDGF released from NPs). Thus, our novel aligned collagen–NP composite fiber has a significant potential to be used for tendon tissue engineering and regeneration.     

An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture

Fabrication of nanofibrous scaffolds with well-defined architecture mimicking native extracellular matrix analog has significant potentials for many specific tissue engineering and organs regeneration applications. The fabrication of aligned collagen nanofibrous scaffolds by electrospinning was described in this study. The structure and in vitro properties of these scaffolds were compared with a random collagen scaffold. All the collagen scaffolds were first crosslinked in glutaraldehyde vapor to enhance the biostability and keep the initial nano-scale dimension intact. From in vitro culture of rabbit conjunctiva fibroblast, the aligned scaffold exhibited lower cell adhesion but higher cell proliferation because of the aligned orientation of fibers when compared with the random scaffold. And the alignment of the fibers may control cell orientation and strengthen the interaction between the cell body and the fibers in the longitudinal direction of the fibers.     

The effect of various denier capillary channel polymer fibers on the alignment of NHDF cells and type I collagen

If tissue engineers are to successfully repair and regenerate native tendons and ligaments, it will be essential to implement contact guidance to induce cellular and type I collagen alignment to replicate the native structure. Capillary channel polymer (CC-P) fibers fabricated by melt-extrusion have aligned micrometer scale surface channels that may serve the goal of achieving biomimetic, physical templates for ligament growth and regeneration. Previous work characterizing the behavior of normal human dermal fibroblasts (NHDF), on the 19 denier per filament (dpf) CC-P fibers, demonstrated a need for improved cellular and type I collagen alignment. Therefore, 5 and 9 dpf CC-P fibers were manufactured to determine whether their channel dimensions would achieve greater alignment. A 29 dpf CC-P fiber was also examined to determine whether cellular guidance could still be achieved within the larger dimensions of the fiber's channels. The 9 dpf CC-P fiber appeared to approach the topographical constraints necessary to induce the cellular and type I collagen architecture that most closely mirrored that of native ACL tissue. This work demonstrated that the novel cross-section of the CC-P fiber geometry could approach the necessary surface topography to align NHDF cells along the longitudinal axis of each fiber.     

Electrospun bio-composite P(LLA-CL)/collagen I/collagen III scaffolds for nerve tissue engineering

One of the biggest challenges in peripheral nerve tissue engineering is to create an artificial nerve graft that could mimic the extracellular matrix (ECM) and assist in nerve regeneration. Bio-composite nanofibrous scaffolds made from synthetic and natural polymeric blends provide suitable substrate for tissue engineering and it can be used as nerve guides eliminating the need of autologous nerve grafts. Nanotopography or orientation of the fibers within the scaffolds greatly influences the nerve cell morphology and outgrowth, and the alignment of the fibers ensures better contact guidance of the cells. In this study, poly (L-lactic acid)-co-poly(ε-caprolactone) or P(LLA-CL), collagen I and collagen III are utilized for the fabrication of nanofibers of different compositions and orientations (random and aligned) by electrospinning. The morphology, mechanical, physical, and chemical properties of the electrospun scaffolds along with their biocompatibility using C17.2 nerve stem cells are studied to identify the suitable material compositions and topography of the electrospun scaffolds required for peripheral nerve regeneration. Aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds with average diameter of 253 ± 102 nm were fabricated and characterized with a tensile strength of 11.59 ± 1.68 MPa. Cell proliferation studies showed 22% increase in cell proliferation on aligned P(LLA-CL)/collagen I/collagen III scaffolds compared with aligned pure P(LLA-CL) scaffolds. Results of our in vitro cell proliferation, cell-scaffold interaction, and neurofilament protein expression studies demonstrated that the electrospun aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds mimic more closely towards the ECM of nerve and have great potential as a substrate for accelerated regeneration of the nerve.     

The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells

Corneal stroma is an avascular connective tissue characterized by layers of highly organized parallel collagen fibrils, mono-disperse in diameter with uniform local interfibrillar spacing. Reproducing this level of structure on a nano- and micro-scale may be essential to engineer corneal tissue with strength and transparency similar to that of native cornea. A substrate of aligned poly(ester urethane) urea (PEUU) fibers, 165 ± 55 nm in diameter, induced alignment of cultured human corneal stromal stem cells (hCSSCs) which elaborated a dense collagenous matrix, 8-10 μm in thickness, deposited on the PEUU substratum. This matrix contained collagen fibrils with uniform diameter and regular interfibrillar spacing, exhibiting global parallel alignment similar to that of native stroma. The cells expressed high levels of gene products unique to keratocytes. hCSSCs cultured on PEUU fibers of random orientation or on a cast film of PEUU also differentiated to keratocytes and produced abundant matrix, but lacked matrix organization. These results demonstrate the importance of topographic cues in instructing organization of the transparent connective tissue of the corneal stroma by differentiated keratocytes. This important information will help with design of biomaterials for a bottom-up strategy to bioengineer spatially complex, collagen-based nano-structured constructs for corneal repair and regeneration.     

Mechanisms of Interstitial Flow-Induced Remodeling of Fibroblast–Collagen Cultures

Interstitial fluid flow, critical for macromolecular transport, was recently shown to drive fibroblast differentiation and perpendicular cell and matrix alignment in 3D collagen cultures. Here we explore the mechanisms underlying this flow-induced cell and collagen alignment. Cell and matrix alignment was assessed from 3D confocal reflectance stacks using a Fast Fourier Transform method. We found that human dermal and lung fibroblasts align perpendicular to flow in the range of 5–13 μm/s (0.1–0.3 dyn/cm²) in collagen; however, neither cells nor matrix fibers align in fibrin cultures, which unlike collagen, is covalently cross-linked and generally degraded by cell fibrinolysis. We also found that even acellular collagen matrices align weakly upon exposure to flow. Matrix alignment begins within 12 h of flow onset and continues, along with cell alignment, over 48 h. Together, these data suggest that interstitial flow first induces collagen fiber alignment, providing contact guidance for the cells to orient along the aligned matrix; later, the aligned cells further remodel and align their surrounding matrix fibers. These findings help elucidate the effects of interstitial flow on cells in matrices and have relevance physiologically in tissue remodeling and in tissue engineering applications.     

Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning

Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured tissues. Important cues in this microenvironment are the chemical, mechanical and spatial arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from relatively low production rates and poor control of fiber alignment without substantial modifications to the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via high-voltage electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun fibers retain greater protein content on the surface and are also fabricated at a higher production rate compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity, and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support cellular alignment, maturation and self-organization. The hybrid nanofiber constructs fabricated by RJS therefore have the potential to be used as scaffold material for a wide variety of biological tissues and organs, as an alternative to electrospinning.     

Biomaterial guides for lymphatic endothelial cell alignment and migration

Axillary dissection during breast cancer surgery produces extensive lymphatic vessel damage that often leads to lifelong secondary lymphedema of the arm. We have developed a biodegradable material conduit for lymphatic vessel reconstruction where fibers electrospun along the conduit lumen promote endothelial cell alignment and migration in vitro. The diameter and density of the electrospun fibers were optimized for cell migration and direction on two-dimensional substrates by seeding human lymphatic endothelial cells (LECs) onto aligned fibers of varying diameters and densities, randomly oriented fibers, and film substrates with no fibers. We found that LECs became aligned in the fiber direction, with cells seeded on the randomly oriented fibers becoming oriented in random directions, whereas cells seeded on the highly aligned fibers became highly aligned. Cell migration was dependent upon fiber alignment and density, with optimal migration found on 1300 nm diameter aligned fibers of low density. Blood endothelial cells seeded on the fibers exhibited similar behavior as the LECs. Fiber alignment was preserved upon rolling the two-dimensional substrate into the tubular geometry of a lymphatic vessel. The data suggest that aligned electrospun fibers may promote endothelial migration across the conduit in a manner that is independent of lymphatic growth factors.     

Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast

The effects of fiber alignment and direction of mechanical stimuli on the ECM generation of human ligament fibroblast (HLF) were assessed. The nanofiber matrix was fabricated using electrospinning technique. To align the nanofibers, a rotating target was used. The HLFs on the aligned nanofibers were spindle-shaped and oriented in the direction of the nanofibers. The degree of ECM production was evaluated by comparing the amount of collagen on aligned and randomly oriented structures. Significantly more collagen was synthesized on aligned nanofiber sheets, although the proliferation did not differ significantly. This suggests that the spindle-shape observable in intact ligaments is preferable in producing ECM. To evaluate the effect of strain direction on the ECM production, HLFs were seeded on parallel aligned, vertically aligned to the strain direction, and randomly oriented nanofiber sheets attached to Flexcell plates. After a 48-h culture, 5% uniaxial strain was applied for 24h at a frequency of 12 cycles/min. The amounts of collagen produced were measured 2 days after halting the strain application. The HLFs were more sensitive to strain in the longitudinal direction. In conclusion, the aligned nanofiber scaffold used in this study constitutes a promising base material for tissue-engineered ligament in that it provides more preferable biomimetic structure, along with proper mechanical environment.     

Continuous cyclic stretch induces osteoblast alignment and formation of anisotropic collagen fiber matrix

Bone tissue geometry shows a highly anisotropic architecture, which is derived from its genetic regulation and mechanical environment. Osteoblasts are responsible not only for bone formation, through the secretion of collagen type I, but also for sensing the mechanical stimuli due to bone surface strain. Mechanotransduction by osteoblasts is therefore considered one of the regulators of anisotropic bone tissue morphogenesis. The orientation of osteoblasts and the secreted collagen matrix was successfully regulated by applying a continuous mechanical stress on osteoblasts for a long period. Under a continuous cyclic stretch of 4% magnitude at a rate of 2 cycles min^?1, osteoblasts reoriented their actin stress fibers in the direction that minimizes the strain applied to them. Extended culture of up to 2 weeks resulted in the formation of collagen fibers in the extracellular spaces, and the preferred orientation of these fibers was parallel to the direction of cell elongation. To the best of our knowledge, this is the first report to establish anisotropic bone matrix architecture following the alignment of osteoblasts under mechanical stimuli for long-term cultivation.     

Quantitative analysis of cell adhesion on aligned micro- and nanofibers

In this study, we quantitatively analyzed the affinity of cell adhesion to aligned nanofibers composed of composites of poly(glycolic acid) (PGA) and collagen. Electrospun composite fibers were fabricated at various PGA/collagen weight mixing ratio (7, 18, 40, 67, and 86%) to generate fibers that ranged in diameter from 10 mum to 500 nm. Scanning electron microscopy (SEM) observation revealed that the PGA/collagen fibers were long and uniformly aligned, irrespective of the PGA/collagen weight mixing ratio. In addition, it was observed that a significantly higher number of NIH3T3 fibroblasts adhered to nanofibers with smaller diameters in comparison to fibers with larger diameters. The highest affinity of cell adhesion was observed in the PGA/collagen fibers with diameter of 500 nm and PGA/collagen weight mixing ratio of 40%. Furthermore, the adherent cells were more elongated on fibers with smaller diameters. Thus, based on the results here, PGA/collagen composite fibers are suitable for tissue culture studies and provide an attractive material for tissue engineering applications.     

Pattern of collagen fiber orientation in the ovine calcaneal shaft and its relation to locomotor-induced strain

Background: Gebhardt (1905. Arch. Entwickl. Org., 20:187–322) originated the hypothesis that the direction of collagen fibers in bone is a structural response to the type of mechanical load to which the bone is subjected. He proposed that collagen fibers aligned parallel to the loading axis are best suited to withstand tensile strain, whereas fibers oriented perpendicular to the loading axis are best able to resist compressive strain. Research comparing load patterns with fiber alignment in bone have tended to support Gebhardt's hypothesis. The aim of the present study is to further test this hypothesis by assessing the correspondence between the distribution of strain and the distribution of collagen fiber orientation in a bone that is subjected to compound loading (i.e., both tension and compression at different phases during the loading cycle). The ovine calcaneum was selected to meet this criterion. Methods: Calcaneum surface strain distributions were obtained from experimental results reported by Lanyon (1973. J. Biomech. 6:41–49). Histological sections of the calcaneal shaft were prepared and observed using circularly polorized light (CPL) microscopy to determine the distribution of collagen fiber alignment. The observed alignment pattern was then compared with the predicted pattern based on Gebhardt's hypothesis. Results: Contrary to previous studies, our findings show no clear correspondence between the strain type of greatest magnitude and the direction of collagen fibers. Areas of bone characterized by high compression and low tension showed predominantly longitudinal collagen alignment (contra to Gebhardt). Conclusions: It is argued that even small magnitudes of tension operating on local areas of bone may be sufficient to induced collagen alignment favorable to this type of strain, even when greater magnitudes of compressive strain are acting on the same bone volume. © 1995 Wiley-Liss, Inc.     

Quantitative analysis of osteoblast-like cells (MG63) morphology on nanogrooved substrata with various groove and ridge dimensions

Nanotextured silicon substrata with parallel ridges separated by grooves with equal width from 90 to 500 nm, were fabricated by electron beam lithography and dry etching techniques. Osteoblast-like cells, MG-63, were cultured on the sterilized nanopatterned substrata for 4 or 24 h, and then imaged by scanning electron microscopy. The influence of substrate topography on cell morphology was analyzed by image software. We found the initially cells spread faster on the nanopatterned surfaces than on the flat surface, suggesting that surface anisotropic feature facilitates initial cell extension along its direction. However, because of inhibition of cell lateral expansion across nanogrooved surfaces, the cells on the nanogrooved surface did not further expand laterally, and cell spreading area was less than that on the flat surface after 24 h of incubation. Cells elongated and aligned along the direction of grooves on all the nanopatterned substrata. Furthermore, fluorescence staining of cell nuclei indicated that the nuclei of the cells cultured on the nanopatterned surfaces also displayed a more elongated and aligned morphology along the direction of the grooves. Since cell shape and orientation influence cell functions and alignment of extracellular matrix secreted by cells, our results may provide the information regarding responses of osteoblasts to the nanostructure of collagen fibrils, and benefit bone tissue engineering and surface design of orthopedic implants.     

Microfluidic alignment of collagen fibers for in vitro cell culture

Three dimensional gels of aligned collagen fibers were patterned in vitro using microfluidic channels. Collagen fiber orientation plays an important role in cell signaling for many tissues in vivo, but alignment has been difficult to realize in vitro. For microfluidic collagen fiber alignment, collagen solution was allowed to polymerize inside polydimethyl siloxane (PDMS) channels ranging from 10–400 μm in width. Collagen fiber orientation increased with smaller channel width, averaging 12 ± 6 degrees from parallel for channels between 10 and 100 μm in width. In these channels 20–40% of the fibers were within 5 degrees of the channel axis. Bovine aortic endothelial cells expressing GFP-tubulin were cultured on aligned collagen substrate and found to stretch in the direction of the fibers. The use of artificially aligned collagen gels could be applied to the study of cell movement, signaling, growth, and differentiation.     

Osteogenic Differentiation of Marrow Stromal Cells on Random and Aligned Electrospun Poly(l-lactide) Nanofibers

The fibrillar structure and sub-micron diameter of electrospun nanofibers can be used to reproduce the morphology and structure of the natural extracellular matrix (ECM). The objective of this work was to investigate the effect of fiber alignment on osteogenic differentiation of bone marrow stromal (BMS) cells. Random and aligned poly(l-lactide) (PLLA) nanofibers were produced by collecting the spun fibers on a stationary plate and a rotating wheel, respectively, as the ground electrode. Morphology and alignment of the BMS cells seeded on the fibers were characterized by SEM. The effect of fiber orientation on osteogenic differentiation of BMS cells was determined by measuring alkaline phosphatase (ALPase) activity, calcium content, and mRNA expression levels of osteogenic markers. There was a strong correlation between the fiber and cell distributions for the random (p = 0.16) and aligned (p = 0.81) fibers. Percent deviation from ideal randomness (PDIR) values indicated that cells seeded on the random fibers (PDIR = 6.5%) were likely to be distributed randomly in all directions while cells seeded on the aligned fibers (PDIR = 86%) were highly likely to be aligned with the direction of fibers. BMS cell seeded on random and aligned fibers had similar cell count and ALPase activity with incubation time, but the calcium content on aligned fibers was significantly higher after 21 days compared to that of random fibers (p = 0.003). Osteopontin (OP) and osteocalcin (OC) expression levels of BMS cells on fibers increased with incubation time. However, there was no difference between the expression levels of OP and OC on aligned vs. random fibers. The results indicate that BMS cells aligned in the direction of PLLA fibers to form long cell extensions, and fiber orientation affected the extent of mineralization, but it had no effect on cell proliferation or mRNA expression of osteogenic markers.