Anatomical meniscus construct with zone specific biochemical composition and structural organization.

A PCL/hydrogel construct that would mimic the structural organization, biochemistry and anatomy of meniscus was engineered. The compressive (380 ±â€¯40 kPa) and tensile modulus (18.2 ±â€¯0.9 MPa) of the PCL scaffolds were increased significantly when constructs were printed with a shifted design and circumferential strands mimicking the collagen organization in native tissue (p < 0.05). Presence of circumferentially aligned PCL strands also led to elongation and alignment of the human fibrochondrocytes. Gene expression of the cells in agarose (Ag), gelatin methacrylate (GelMA), and GelMA-Ag hydrogels was significantly higher than that of cells on the PCL scaffolds after a 21-day culture. GelMA exhibited the highest level of collagen type I (COL1A2) mRNA expression, while GelMA-Ag exhibited the highest level of aggrecan (AGG) expression (p < 0.001, compared to PCL). GelMA and GelMA-Ag exhibited a high level of collagen type II (COL2A1) expression (p < 0.05, compared to PCL). Anatomical scaffolds with circumferential PCL strands were impregnated with cell-loaded GelMA in the periphery and GelMA-Ag in the inner region. GelMA and GelMA-Ag hydrogels enhanced the production of COL 1 and COL 2 proteins after a 6-week culture (p < 0.05). COL 1 expression increased gradually towards the outer periphery, while COL 2 expression decreased. We were thus able to engineer an anatomical meniscus with a cartilage-like inner region and fibrocartilage-like outer region.

Square prism micropillars improve osteogenicity of poly(methyl methacrylate) surfaces.

Osteogenicity and osteointegration of materials is one of the key elements of the success of bone implants. Poly(methyl methacrylate) (PMMA) is the basic compound of bone cement and has been widely investigated for other orthopedic applications, but its poor osteointegration and the subsequent loosening of implant material limits its widespread use as bone implants. Micropillar features on substrate surfaces were recently reported to modulate cell behavior through alteration of cell morphology and promotion of osteogenesis. Utilization of this pillar-decorated topography may be an effective approach to enhance osteogenicity of polymeric surfaces. The aim of this study was to investigate the effect of cell morphology on the micropillar features on attachment, proliferation, and osteogenic activity of human osteoblast-like cells. A series of solvent cast PMMA films decorated with 8 µm high square prism micropillars with pillar width and interpillar distances of 4, 8 and 16 µm were prepared from photolithographic templates, and primary human osteoblast-like cells (hOB) isolated from bone fragments were cultured on them. Micropillars increased cell attachment and early proliferation rate compared to unpatterned surfaces, and triggered distinct morphological changes in cell body and nucleus. Surfaces with pillar dimensions and gap width of 4 µm presented the best osteogenic activity. Expression of osteogenic marker genes was upregulated by micropillars, and cells formed bone nodule-like aggregates rich in bone matrix proteins and calcium phosphate. These results indicated that micropillar features enhance osteogenic activity on PMMA films, possibly by triggering morphological changes that promote the osteogenic phenotype of the cells.

Effects of microarchitecture and mechanical properties of 3D microporous PLLA-PLGA scaffolds on fibrochondrocyte and L929 fibroblast behavior.

There are several reports studying cell behavior on surfaces in 2D or in hydrogels in 3D. However, cell behavior in 3D microporous scaffolds has not been investigated extensively. In this study, poly(L-lactic acid)/poly(lactic acid-co-glycolic acid) (PLLA/PLGA)-based microporous scaffolds were used to study the effects of scaffold microarchitecture and mechanical properties on the behavior of two different cell types, human meniscal fibrochondrocytes and L929 mouse fibroblasts. In general, cell attachment, spreading and proliferation rate were mainly regulated by the strut (pore wall) stiffness. Increasing strut stiffness resulted in an increase in L929 fibroblast attachment and a decrease in fibrochondrocyte attachment. L929 fibroblasts tended to get more round as the strut stiffness increased, while fibrochondrocytes tended to get more elongated. Cell migration increased for both cell types with the increasing pore size. Migrating L929 fibroblasts tended to get more round on the stiff scaffolds, while fibrochondrocytes tended to get more round on the soft scaffolds. This study shows that the behavior of cells on 3D microporous scaffolds is mainly regulated by pore size and strut stiffness, and the response of a cell depends on the stiffness of both cells and materials. This study could be useful in designing better scaffolds for tissue engineering applications.

Cartilage tissue engineering on macroporous scaffolds using human tooth germ stem cells.

The main objective was to study cartilage regeneration through differentiation of human tooth germ stem cells (HTGSCs) into chondrocytes on different three-dimensional (3D) scaffolds (PCL, PLLA and PCL-PLLA). Scaffold topographies were studied by scanning electron microscopy and it was found that the scaffolds had interconnected macroporous structures. HTGSCs were isolated from impacted third molar tooth germs of young adult patients and grown for 3 weeks on the scaffolds in chondrogenic differentiation medium. Cell proliferation on the scaffolds was determined by MTS assay and it was observed that all scaffolds supported cell proliferation. Immunostaining was carried out for morphological and differentiation analyses. Immunohistochemical analyses revealed that the cells attached onto the scaffolds and deposited cartilage-specific extracellular matrix (ECM). Real-time PCR was performed to determine the expression levels of cartilage-specific genes. After 21 days of incubation in cartilage differentiation medium, expression of collagen type II increased only in the cells seeded onto PCL-PLLA blend scaffolds. Similarly, aggrecan expression was the highest on PCL-PLLA scaffolds after 3 weeks. These results suggest that all the scaffolds, and especially PCL-PLLA, were suitable for chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd.

Peripheral nerve conduits: technology update.

Peripheral nerve injury is a worldwide clinical problem which could lead to loss of neuronal communication along sensory and motor nerves between the central nervous system (CNS) and the peripheral organs and impairs the quality of life of a patient. The primary requirement for the treatment of complete lesions is a tension-free, end-to-end repair. When end-to-end repair is not possible, peripheral nerve grafts or nerve conduits are used. The limited availability of autografts, and drawbacks of the allografts and xenografts like immunological reactions, forced the researchers to investigate and develop alternative approaches, mainly nerve conduits. In this review, recent information on the various types of conduit materials (made of biological and synthetic polymers) and designs (tubular, fibrous, and matrix type) are being presented.

Construction of a collagen-based, split-thickness cornea substitute.

Tissue-engineered corneas may become a promising alternative to allografts in the treatment of serious cornea defects because of the tunable characteristics of the biomaterials, biomimetic designs, and incorporation of patient's own cells. In this study, collagen foam was coated with a fibrous mat to mimic the stromal layer and the Bowman's layer. The stromal layer substitute was made of N-ethyl-N-(3-dimethyl aminopropyl)carbodiimide/N-hydroxysuccinimide-cross-linked collagen-chondroitin sulfate foam and seeded with primary human corneal keratocytes (HK). Retinal pigment epithelium (RPE) cells served as the epithelial layer after seeding on a dehydrothermally cross-linked collagen type I fibrous mat deposited directly on top of the foams by electrospinning. The physical characterization and the in vitro studies showed that the designed cornea replacement was suitable for cell attachment and growth, and co-culture of the two cell types induced more extracellular matrix (ECM) deposition than the single cell-seeded constructs. The fiber layer was shown to be successful in separating the HK and RPE cells, and still allowed them to maintain cell-cell communication as the increase in ECM deposition and the maintenance of the high transparency (~80%) suggested. This split-thickness corneal substitute was also shown to be readily suturable without any major tears at the end of a short co-culture of 30 days.

In vitro and transdermal penetration of PHBV micro/nanoparticles.

The purpose of this study was to develop micro and nano sized drug carriers from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and study the cell and skin penetration of these particles. PHBV micro/nanospheres were prepared by o/w emulsion method and were stained with a fluorescent dye, Nile Red. The particles were fractionated by centrifugation to produce different sized populations. Topography was studied by SEM and average particle size and its distribution were determined with particle sizer. Cell viability assay (MTT) was carried out using L929 fibroblastic cell line, and particle penetration into the cells were studied. Transdermal permeation of PHBV micro/nanospheres and tissue reaction were studied using a BALB/c mouse model. Skin response was evaluated histologically and amount of PHBV in skin was determined by gas chromatography-mass spectrometry. The average diameters of the PHBV micro/nanosphere batches were found to be 1.9 µm, 426 and 166 nm. Polydispersity indices showed that the size distribution of micro sized particles was broader than the smaller ones. In vitro studies showed that the cells had a normal growth trend. MTT showed no signs of particle toxicity. The 426 and 166 nm sized PHBV spheres were seen to penetrate the cell membrane. The histological sections revealed no adverse effects. In view of this data nano and micro sized PHBV particles appeared to have potential to serve as topical and transdermal drug delivery carriers for use on aged or damaged skin or in cases of skin diseases such as psoriasis, and may even be used in gene transfer to cells.

Multiwalled CNT-pHEMA composite conduit for peripheral nerve repair.

A nerve conduit is designed to improve peripheral nerve regeneration by providing guidance to the nerve cells. Conductivity of such guides is reported to enhance this process. In the current study, a nerve guide was constructed from poly(2-hydroxyethyl methacrylate) (pHEMA), which was loaded with multiwalled carbon nanotubes (mwCNT) to introduce conductivity. PHEMA hydrogels were designed to have a porous structure to facilitate the transportation of the compounds needed for cell nutrition and growth and also for waste removal. We showed that when loaded with relatively high concentrations of mwCNTs (6%, w/w in hydrogels), the pHEMA guide was more conductive and more hydrophobic than pristine pHEMA hydrogel. The mechanical properties of the composites were better when they carried mwCNT. Elastic modulus of 6% mwCNT loaded pHEMA was twofold higher (0.32 ± 0.06 MPa) and similar to that of the soft tissues. Electrical conductivity was significantly improved (11.4-fold) from 7 × 10(-3) Ω(-1).cm(-1) (pHEMA) to 8.0 × 10(-2) Ω(-1).cm(-1) (6% mwCNT loaded pHEMA). On application of electrical potential, the SHSY5Y neuroblastoma cells seeded on mwCNTs carrying pHEMA maintained their viability, whereas those on pure pHEMA could not, indicating that mwCNT helped conduct electricity and make them more suitable as nerve conduits.

An in vivo study on the effect of scaffold geometry and growth factor release on the healing of bone defects.

The hypothesis of this study was that the extent of bone regeneration could be enhanced by using scaffolds with appropriate geometry, and that such an effect could be further increased by mimicking the natural timing of appearance of bone morphogenetic proteins BMP-2 and BMP-7 after fracture. Bioplotted poly(ε-caprolactone) (PCL) disks with four different fibre organizations were used to study the effect of 3D scaffold architecture on the healing of bone defects in a rat pelvis model. Moreover, one PCL construct was further modified by introducing a nanoparticulate sequential BMP-2/BMP-7 delivery system into this scaffold. Scaffolds and functionalized construct along with free nanocapsules were implanted using a rat iliac crest defect model. Six weeks post-implantation, the defects were evaluated by CT scan and histology. Analysis revealed that the basic architecture, having the highest pore volume for tissue ingrowth, presented the highest bone formation as determined by the bone mineral density (BMD) within the defect (144.2 ± 7.1); about four-fold higher than that of the empty defect (34.9 ± 10.7). It also showed the highest histological analysis scores with a high amount of bone formation within the defect, within the scaffold pores and along the outer surfaces of the scaffold. The basic scaffold carrying the BMP-2/BMP-7 delivery system showed significantly higher bone formation than the growth factor-free basic scaffold at 6 weeks (BMD 206.8 ± 15.7). Histological analysis also revealed new bone formation in close to or in direct contact with the construct interface. This study indicates the importance of open and interconnecting pore geometry on the better healing of bone defects, and that this effect could be further increased by supplying growth factors, as is the case in nature.

Porous Agarose-Based Semi-IPN Hydrogels: Characterization and Cell Affinity Studies.

Hydrogels are frequently considered for medical applications due to the ease of preparation in different forms and high water content that makes them comparable to natural tissues. However, these general properties are not sufficient to make any hydrogel suitable for cell attachment and growth which are necessary for their use in tissue regeneration. Besides, the high water content makes the hydrogels mechanically weak. The formation of semi-interpenetrating networks (semi-IPNs) can be used in attempts to enhance physical, mechanical and thermal properties. In this study, semi-IPNs of agarose were prepared with chitosan and alginate, two polyelectrolytes that are positively and negatively charged under physiological conditions, respectively. Zeta potential was used to confirm the formation of charged hydrogels. All hydrogels had ultimate compression strengths in the range of 91-210 Pa where the value for pure agarose was about 103 Pa. Chitosan increased the compressive strength about two folds whereas the alginate had opposite effects. The amount of strongly bound water present in the hydrogels were estimated from TGA and DSC analysis and the highest value was found for alginate-agarose hydrogels as about 15%. The attachment and the migration of L929 fibroblasts were monitored in vitro using the MTS assay and confocal microscopy. The highest cell proliferation and penetration were observed for positively charged chitosan-agarose semi-IPN hydrogels.

Development of porous chitosan-gelatin/hydroxyapatite composite scaffolds for hard tissue-engineering applications.

Composite scaffolds prepared from natural polymers and hydroxyapatite (HA) are expected to have enhanced osteoconductive properties and as a result gained much attention in recent years for use in bone tissue-engineering applications. Although there are various natural polymers available for this purpose, chitosan (C) and gelatin (G) are commonly studied because of their inherent properties. The aim of this study was to prepare three-dimensional (3D) scaffolds using these two natural polymers and to add either non-sintered hydroxyapatite (nsHA) or sintered hydroxyapatite (sHA) to compare their influence on physical, chemical and mechanical properties of the scaffolds and on their affinities towards Saos-2 cells. For this purpose, nsHA and sHA were synthesized and characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM) and particle size analyses. Then nsHA and sHA particles, with average sizes of 16 µm and 6 µm, respectively, were added to the solutions of C and G during the preparation step and the resultant 3D scaffolds were characterized. Compression tests indicated that presence of nsHA or sHA increased the Young's modulus and compressive strength of the scaffolds, and the values were very similar to those of human spongy bone. MTS assays, confocal microscopy and SEM analysis showed that cell attachment and proliferation were higher on C-G/sHA composite scaffolds compared to the other scaffolds. It was shown that the scaffolds prepared from chitosan, gelatin and HA are appropriate cell carriers for bone tissue engineering, especially those with sHA incorporated.

Microfabrication of PDLLA scaffolds.

This study aimed to comprehend the potentialities of the microfabrication to produce tissue-engineering scaffolds. Structures presenting homogeneously distributed pores of size 100 and 200 µm were fabricated through layer-by-layer deposition of filaments of poly(D,L-lactic acid) (PDLLA) prepared from dichloromethane/dimethylformamide solutions. Rheological tests on the solution and molecular weight distributions of PDLLA, solvent cast films and microfabricated scaffolds were performed to determine which material conditions are optimal for the microfabricated system and to identify any possible material modification induced by the process. In vitro qualitative preliminary cell culture studies were conducted using MG63 osteoblast cell lines after assuring the non-cytotoxicity of the scaffold material by the lactate dehydrogenase in vitro toxicology assay; biological evaluations were initially performed using scaffolds with the smaller (100 µm) pore size. Scanning electron microscopy imaging was used to determine cell morphology distribution. A second cell culture test was performed, using the scaffold with the higher (200 µm) porosity. Confocal laser microscopy (CLM) was utilized to examine cell morphology and growth behaviour. Cellular metabolic activity and viability were also examined using Alamar Blue assay and further verifications were performed using CLM. Cell culture studies indicated homogeneous distribution, high viability and metabolic activity. Pore dimension affects cell distribution: pores < 100 µm acted as barrier structures for the MG63 osteoblast cell line; penetration inside the matrix was hindered and cells grew on the outer part. Increasing pore size resulted in a more homogeneous cell distribution and penetration of cells inside the structure was achieved.

Both sides nanopatterned tubular collagen scaffolds as tissue-engineered vascular grafts.

Two major requirements for a tissue-engineered vessel are the establishment of a continuous endothelium and adequate mechanical properties. In this study, a novel tubular collagen scaffold possessing nanopatterns in the form of channels (with a 650 nm periodicity) on both sides was designed and examined after seeding and co-culturing with vascular cells. Initially, the exterior of the tube was seeded with human vascular smooth muscle cells (VSMCs), cultured for 14 days, and then human internal thoracic artery endothelial cells (HITAECs) were seeded on the inside of the tube and cultured for a further week. Microscopy revealed that nano-scale patterns could be reproduced on collagen with high fidelity and preserved during incubation in vitro. The VSMCs were circumferentially orientated with the help of these nanopatterns and formed multilayers on the exterior, while HITAECs formed a continuous layer on the interior, as is the case in natural vessels. Both cell types were observed to proliferate and retain their phenotypes in the co-culture.

Design of a 3D aligned myocardial tissue construct from biodegradable polyesters.

The heart does not regenerate new functional tissue when myocardium dies following coronary artery occlusion, or if it is defective. Ventricular restoration involves excising the infarct and replacing it with a cardiac patch to restore the heart to a more healthy condition. The goal of this study was to design and develop a clinically applicable myocardial patch to replace myocardial infarcts and improve long-term heart function. A basic design composed of 3D microfibrous mats that house mesenchymal stem cells (MSCs) was developed from human umbilical cord matrix (Wharton's Jelly) cells aligned in parallel to each other mimicking the native myocardium. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(L-D,L-lactic acid) (P(L-D,L)LA) and poly(glycerol sebacate) (PGS) were blended and electrospun into aligned fiber mats with fiber diameter ranging between 1.10 and 1.25 microm. The micron-sized parallel fibers of the polymer blend were effective in cell alignment and cells have penetrated deep within the mat through the fiber interstices, occupying the whole structure; 8-9 cell layers were obtained. Biodegradable macroporous tubings were introduced to serve as nutrient delivery route. It was possible to create a thick myocardial patch with structure similar to the native tissue and with a capability to grow.

Sequential BMP-2/BMP-7 delivery from polyester nanocapsules.

The aim of this study was to develop a nanosized, controlled growth factor release system to incorporate into tissue engineering scaffolds and thus activate the cells seeded in the scaffold. Nanocapsules of poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were loaded with the bone morphogenetic proteins BMP-2 and BMP-7, respectively, and with bovine serum albumin (BSA), the model protein. BSA-loading efficiency and release kinetics were used to determine the most appropriate nanocapsule pair to achieve the delivery of growth factors in a sequential manner, as occurs in natural processes. BSA-encapsulation efficiency was highest when the polymer concentration used in the preparation of PLGA and PHBV nanocapsules was 10% (w/v) (84.75% and 16.72%, respectively). Release of BSA was faster from PLGA than it was from PHBV. Based on the encapsulation efficiency and release data, 10% PLGA and 10% PHBV nanocapsules were chosen to provide the early BMP-2 and later BMP-7 release, respectively. Simultaneous, sequential delivery and individual release of the BMPs were studied for 7, 14, and 21 days, using rat bone marrow mesenchymal stem cells. Individual BMP-2 release suppressed cell proliferation while providing higher alkaline phosphatase activity with respect to BMP-7. The sequential delivery of BMP-2 and BMP-7 provided slightly lower proliferation than did simultaneous delivery, but the highest alkaline phosphatase activity of all indicated a synergistic effect on the osteogenic differentiation of mesenchymal stem cells caused by the use of the two growth factors in a sequential fashion.

Influence of nanopatterns on endothelial cell adhesion: Enhanced cell retention under shear stress.

In this study, nanopatterned crosslinked films of collagen Type I were seeded with human microvascular endothelial cells and tested for their suitability for vascular tissue engineering. Since the films will be rolled into tubes with concentric layers of collagen, nutrient transfer through the collagen films is quite crucial. Molecular diffusivity through the collagen films, cell viability, cell proliferation and cell retention following shear stress were studied. Cells were seeded onto linearly nanogrooved films (groove widths of 332.5, 500 and 650nm), with the grooves aligned in the direction of flow. The nanopatterns did not affect cell proliferation or initial cell alignment; however, they significantly affected cell retention under fluid flow. While cell retention on unpatterned films was 35+/-10%, it was 75+/-4% on 332.5nm patterned films and even higher, 91+/-5%, on 650nm patterned films. The films were found to have diffusion coefficients of ca. 10(-6)cm(2)s(-1) for O(2) and 4-acetaminophenol, which is comparable to that observed in natural tissues. This constitutes another positive asset of these films for consideration as a scaffold material for vascular tissue engineering.

Nanopatterning of collagen scaffolds improve the mechanical properties of tissue engineered vascular grafts.

Tissue engineered constructs with cells growing in an organized manner have been shown to have improved mechanical properties. This can be especially important when constructing tissues that need to perform under load, such as cardiac and vascular tissue. Enhancement of mechanical properties of tissue engineered vascular grafts via orientation of smooth muscle cells by the help of topographical cues have not been reported yet. In the present study, collagen scaffolds with 650, 500, and 332.5 nm wide nanochannels and ridges were designed and seeded with smooth muscle cells isolated from the human saphenous vein. Cell alignment on the construct was shown by SEM and fluorescence microscopy. The ultimate tensile strength (UTS) and Young's modulus of the scaffolds were determined after 45 and 75 days. Alamar Blue assay was used to determine the number of viable cells on surfaces with different dimensioned patterns. Presence of nanopatterns increased the UTS from 0.55 +/- 0.11 to as much as 1.63 +/- 0.46 MPa, a value within the range of natural arteries and veins. Similarly, Young's modulus values were found to be around 4 MPa, again in the range of natural vessels. The study thus showed that nanopatterns as small as 332.5 nm could align the smooth muscle cells and that alignment significantly improved mechanical properties, indicating that nanopatterned collagen scaffolds have the potential for use in the tissue engineering of small diameter blood vessels.

Nanopatterned collagen tubes for vascular tissue engineering.

Nanopatterned (330 nm wide channels) type I collagen films were prepared by solvent casting on poly(dimethyl siloxane) (PDMS) templates. These films were rolled into tubular constructs and crosslinked. Tubular constructs were incubated under cell culture conditions for 28 days and examined by stereomicroscopy and scanning electron microscopy (SEM) for the integrity of the structure. The nanopatterned films were also seeded with human vascular smooth muscle cells (VSMCs) and examined after immunostaining with fluorescence microscopy and SEM to assess the cell phenotype and alignment on the nanopatterns on the films.

A novel construct as a cell carrier for tissue engineering.

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.

Chemical and topographical modification of PHBV surface to promote osteoblast alignment and confinement.

Proper cell attachment and distribution, and thus stronger association in vivo between a bone implant and native tissue will improve the success of the implant. In this study, the aim was to achieve promotion of attachment and uniform distribution of rat mesenchymal stem cell-derived osteoblasts by introducing chemical and topographical cues on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film surfaces. As the chemical cues, either alkaline phosphatase was covalently immobilized on the film surface to induce deposition of calcium phosphate minerals or fibrinogen was adsorbed to improve cell adhesion. Microgrooves and micropits were introduced on the film surface by negative replication of micropatterned Si wafers. Both chemical cues improved cell attachment and even distribution on the PHBV films, but Fb was more effective especially when combined with the micropatterns. Cell alignment (<10 degrees deviation angle) parallel to chemically modified microgrooves (1, 3, or 8 microm groove width) and on 10 microm-thick Fb lines printed on the unpatterned films was achieved. The cells on unpatterned and 5 microm-deep micropitted films were distributed and oriented randomly. Results of this study proved that microtopographies on PHBV can improve osseointegration when combined with chemical cues, and that microgrooves and cell adhesive protein lines on PHBV can guide selective osteoblast adhesion and alignment.