PHBV Films Research Articles

Surface engineering of PHBV by covalent collagen immobilization to improve cell compatibility

Covalent immobilization of collagen onto poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film was achieved to improve its cell compatibility. Amide groups photografted on PHBV films were initially converted into amine groups through Hofmann degradation and collagen was then chemically bonded to amine groups, consequently forming the amide, amine, and collagen-modified PHBV. The structures of these modified PHBV films were confirmed by ATR-FTIR, XPS, and SEM analyses. Compared with that of PHBV film, surface wettability of the modified PHBV films enhanced remarkably. In particular, water contact angle of the collagen-modified PHBV film decreased from 65.0 degrees to 2.1 degrees within 130 s. Sheep chondrocytes cultured on PHBV and modified PHBV films were evaluated by cell adhesion test, MTT assay, and morphological observation under SEM. Results showed that the collagen-modified PHBV film had better cell adhesion and proliferation than other modified PHBV films and PHBV film. Chondrocytes on the collagen-modified PHBV film adhered through filopodia, spread by cytoplasmic webbing, and formed cells layer earlier than other modified ones, indicating that the collagen-modified PHBV is a promising biomaterial for cartilage tissue engineering.

Superhydrophobicity of PHBV fibrous surface with bead-on-string structure

A poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) fibrous surface with various bead-on-string structures was fabricated by electrospinning. PHBV was electrospun at various concentrations and then CF 4 plasma treatment was employed to further improve the hydrophobicity of the PHBV fiber surfaces. The surface morphology of the electrospun PHBV mats was observed by scanning electron microscopy (SEM). The surface properties were characterized by water contact angle (WCA) measurements and X-ray photoelectron spectroscopy (XPS). The surface morphology of the electrospun PHBV fibrous mats with the bead-son-string structure varied with the solution concentration. The WCA of all of the electrospun PHBV mats was higher than that of the PHBV film. In particular, a very rough fiber surface including porous beads was observed when PHBV was electrospun from the solution with a concentration of 26 wt%. Also, its WCA further increased from 141° to 158° after CF 4 plasma treatment for 150 s. PHBV can be rendered superhydrophobic by controlling the surface morphology and surface energy, which can be achieved by adjusting the electrospinning and plasma treatment conditions.

Photografting polymerization of polyacrylamide on poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) films. II. Wettability and crystallization behaviors of poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)‐graft‐polyacrylamide films

AbstractThe wettability and crystallization behaviors of poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV)‐graft‐polyacrylamide (PAM) films were studied. X‐ray photoelectron spectroscopy analyses illustrated that about 62 atom % of the total polar functionalities on the grafted film with 17% grafting percentage (GP) was amide groups. Wide‐angle X‐ray diffraction results suggest that grafted PAM induced defects in PHBV crystals and influenced their crystal structure. Differential scanning calorimetry (DSC) spectra showed the two melting regions, 60–90 and 145–170°C, of the imperfect PHBV crystals of the grafted films. Grafted PAM could suppress the recrystallization of PHBV, which was consistent with the polarizing optical microscopy results, in which the maximum PHBV spherulite diameter decreased from 350 μm for the PHBV film to 50 μm for the film with 53% GP. In addition, DSC studies revealed that the crystallinity of the grafted films decreased with increasing GP, which facilitated the diffusion of water into the films. The water contact angle of grafted films decreased and the water‐swelling percentage increased as GP went up. These results demonstrate the potential of PHBV‐g‐PAM for wettable surface constructs in tissue engineering applications. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2008

Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydrolysis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 °C, 1% substrate concentration and in the presence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified fromS. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO2 production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities ofS. kashmirense AF1.

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.

Development of Rapid Cell Recovery System Using Temperature-Responsive Nanofiber Surfaces

PHBV ultrafine fibers were fabricated by electrospinning process. Electrospun PHBV fiber structures revealed randomly aligned fibers with average diameter of 400 nm. PIPAAm was grafted on the surface of PHBV nanofibrous mat by electron beam irradiation. PIPAAm-grafted PHBV mats were determined by ATR-FTIR and ESCA. Water contact angles were determined by a sessile drop method at 20 and 37. To examine the tissue compatibility, human fibroblasts were evenly seeded onto PIPAAm-grafted PHBV mat and cast film, ungrafted PHBV mat and film. Attached and spread fibroblasts on nanofibrous mat were proliferated more rapidly than that of flat film surface. Initial cell attachment on PIPAAm-grafted surfaces was higher than ungrafted surfaces. The surface property changed to hydrophilic by PIPAAm graft, which increased initial cell attachment. Detachment of single cells from PIPAAm-grafted PHBV matrixes was measured by low temperature treatment after incubation at 37. Cultured cells were rapidly detached from PIPAAm-grafted PHBV mat compared with film. With porous mats, the water molecules easily reach to grafted PIPAAm from underneath and peripheral to the attached cells, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cells.

Investigating the structure–property relationship of bacterial PHA block copolymers

Mechanical testing of solvent cast films consisting of short-chain-length (SCL) polyhydroxyalkanoate (PHA) films suggested that films consisting of block copolymers retained more elasticity over time with respect to films of similar random copolymers of comparable composition. Two experimental techniques, wide angle X-ray scattering (WAXS) and uniaxial extension, were used to quantitatively investigate the structure–property relationship of bacterially synthesized PHA block copolymers of poly(3-hydroxybutyrate) (PHB) homopolymer and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) random copolymer (PHBV) segments. Uniaxial testing experiments yielded the Young's modulus, ultimate tensile strength, and the elongation until fracture of the films. Percent crystallinity was determined by deconvolution of amorphous and crystalline scattering peaks obtained from WAXS. Two PHBV films containing either 8% 3-hydroxyvalerate monomer (3HV) or 29% 3HV exhibited a quick transition to brittle behavior, decreasing to less than 20% percent elongation at fracture within a few days after annealing. Conversely, the block copolymer samples remained higher than 100% elongation at fracture a full 3 months after annealing. Because block copolymers covalently link polymers that would otherwise form thermodynamically separate phases, the rates and degrees of crystallization of the block copolymers are less than the random copolymer samples. These differences translate into materials that extend the property space of biologically synthesized SCL PHA.

In Vitro Biocompatibility of Schwann Cells on Surfaces of Biocompatible Polymeric Electrospun Fibrous and Solution-Cast Film Scaffolds

The in vitro responses of Schwann cells (RT4-D6P2T, a schwannoma cell line derived from a chemically induced rat peripheral neurotumor) on various types of electrospun fibrous scaffolds of some commercially available biocompatible and biodegradable polymers, i.e., poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polycaprolactone (PCL), poly(l-lactic acid) (PLLA), and chitosan (CS), were reported in comparison with those of the cells on corresponding solution-cast film scaffolds as well as on a tissue-culture polystyrene plate (TCPS), used as the positive control. At 24 h after cell seeding, the viability of the attached cells on the various substrates could be ranked as follows: PCL film > TCPS > PCL fibrous > PLLA fibrous > PHBV film > CS fibrous approximately CS film approximately PLLA film > PHB film > PHBV fibrous > PHB fibrous. At day 3 of cell culture, the viability of the proliferated cells on the various substrates could be ranked as follows: TCPS > PHBV film > PLLA film > PCL film > PLLA fibrous > PHB film approximately PCL fibrous > CS fibrous > CS film > PHB fibrous > PHBV fibrous. At approximately 8 h after cell seeding, the cells on the flat surfaces of all of the film scaffolds and that of the PCL nanofibrous scaffold appeared in their characteristic spindle shape, while those on the surfaces of the PHB, PHBV, and PLLA macrofibrous scaffolds also appeared in their characteristic spindle shape, but with the cells being able to penetrate to the inner side of the scaffolds.

Fluorinated-plasma coating on polyhydroxyalcanoate PHBV: Effect on the biodegradation

Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) was covered with an hydrophobic layer from plasma polymerization of tetradecafluorohexane, octadecafluorooctane, 3,3,4,4,5,5,6,6,6-nonafluoro-hex-1-ene and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-oct-1-ene. The water contact angle increased from 74° for untreated films to 98° for the treated films and the surface energy decreased from 40.9 to 18.8 mJ m −2. XPS analysis showed the introduction of 54% fluorine and 3–7% oxygen, the binding energies were assigned to chemically differently bonded carbon atoms. CF 2/CF 3 molar ratio in plasma layers was lower than that in the monomers due to molecular fragmentation, however the extent of monomer structure retention in PFH, PFO saturated chains is higher than in NFH, TDFO unsaturated chains. Biodegradation tests under aerobic conditions showed that the fluorinated plasma layer inhibited the biodegradation of the PHBV film underneath.

Electrospinning of microbial polyester for cell culture

Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture.

Photografting polymerization of polyacrylamide on PHBV films (I)

Photografting polymerization of polyacrylamide (PAM) onto poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films using benzophenone as photoinitiator was studied. The morphology and structure of the grafted PHBV film were characterized by Fourier transformed infrared spectroscopy (FTIR) with attenuated total reflectance (ATR) and scanning electron microscope (SEM) with energy dispersive X-ray spectrometer (EDX). The grafting percentage and grafting efficiency of the grafted PHBV film went up with the increase of acrylamide concentration and irradiation time. It was observed that photografting polymerization of PAM was not only limited to the film surface, but also in situ occurred inside the film to form the pore microstructure. Sheep bone marrow stromal cell studies showed that MSCs cells attachment efficiency on the grafted PHBV films increased and cells grew well. These results demonstrated the potentiality of PAM-photografting PHBV in medical applications. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104, 4088–4095, 2007

Investigations into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) surface properties causing delayed osteoblast growth

Osteoblast proliferation is sensitive to material surface properties. In this study, the proliferation of MC3T3 E1-S14 osteoblastic cells on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films with different surface characteristics was investigated with the aim of evaluating the cause of a lag in cell growth previously observed. The solvent-cast films were prepared using three different solvents/solvent mixtures which produced PHBV films with both a rough (at the air interface) and smooth (at the glass interface) surface. Investigation of the surface roughness by scanning electron and scanning probe microscopy revealed that the surfaces had features that were different in both average lateral size and average amplitude (R a 20–200 nm). Water contact angles showed that all surfaces were hydrophobic in nature ( A in the range 69–82°). The lateral distribution of surface crystallinity of the films was evaluated by use of micro-attenuated total reflectance Fourier transform infrared (ATR–FT-IR) by determining the surface crystallinity index (CI) which was found to differ between samples. MC3T3-E1-S14 osteoblasts were cultured on the six surfaces and proliferation was determined. After 2 days, cell proliferation on all surfaces was significantly less than on the control substrate; however, after 4 days cell proliferation was optimal on three surfaces. It was concluded that the initial lag on all substrates was due to the hydrophobic nature of the substrates. The ability of the cells to recover on the materials was attributed to the degree of heterogeneity of the crystallinity and surface roughness: samples with a roughness of 80 nm were found to support cell proliferation. In addition, the lateral surface features influenced the proliferation of osteoblasts on the PHBV film surface.

In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) fiber mats

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.

Reduction of the in vitro pro-inflammatory response by macrophages to poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

This study evaluates the pro-inflammatory response to the thermoplastic biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through the analysis of cellular responses in vitro. The murine macrophage RAW264.7 cell line was cultured on solvent cast PHBV films, which was found to induce pro-inflammatory activity that required direct contact between the material and the macrophages. The identity of the pro-inflammatory stimulus was determined by culturing bone marrow-derived macrophages from bacterial lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice and CpG non-responsive TLR9-/- mice on PHBV. The lack of a pro-inflammatory response by the C3H/HeJ cells indicates that the pro-inflammatory agent present within PHBV is predominately LPS while the TLR9-/- macrophages confirmed that CpG-containing bacterial DNA is unlikely to contribute to the activity. A series of purification procedures was evaluated and one procedure was developed that utilized hydrogen peroxide treatment in solution. The optimized purification was found to substantially reduce the pro-inflammatory response to PHBV without adversely affecting either the molecular structure or molecular weight of the material thereby rendering it more amenable for use as a biomaterial in vivo.

Thermoreversible gelation of biodegradable polyester (PHBV) in toluene

In present paper we investigate thermoreversible gelation of biodegradable polyester poly (3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) in toluene. Hot PHBV solutions became transparent gels after cooling to room temperature. This physical gelation process was followed by light scattering and viscosity measurements for solutions of different PHBV concentrations. It has been found that gelation temperature increases with increasing polymer concentration in toluene. PHBV films have been prepared by gelation process followed by solvent removal on solid substrates. It has been demonstrated that PHBV concentration in the solution influences the surface morphology of obtained films. Homogeneous PHBV films with increased surface roughness can be obtained by means of developed technique. Hydrolytic degradation studies indicate that surface morphology of obtained PHBV layers changes considerably with degradation time.

Improvement in hydrophilicity of PHBV films by plasma treatment

Poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) films were prepared by the solvent cast method. Oxygen and nitrogen plasma treatment were used to improve the hydrophilicity of the surface of PHBV films. The surface properties were characterized by contact angle measurement and X-ray photoelectron spectroscopy (XPS). The water contact angle of PHBV films decreased after plasma treatment, which suggested that the surfaces became more hydrophilic. The effects of exposure time, plasma generating power, and chamber pressure on water contact angle were investigated. Although the decrease of contact angle with both plasma treatments was similar, the change of PHBV surface structure was different. XPS analysis showed that the oxygen content and the ratio of O/C increased markedly after oxygen plasma treatment, while after nitrogen plasma treatment, the surface was enriched with nitrogen atoms. Furthermore, it was found that C-O and C-C bonds were broken with both plasma treatment, while different new bonds were formed, that is, COOH (for oxygen plasma), and C-N, C=N, and amide bonds (for nitrogen plasma) have been generated after plasma irradiation. Dog bone marrow stromal cell culture studies showed that cells grew better on the plasma treated surface. These results demonstrate potentiality of this method for grafting useful component in future tissue-engineering applications.

Bone cell viability on methacrylic acid grafted and collagen immobilized porous poly(3‐hydroxybutrate‐co‐3‐hydroxyvalerate)

AbstractPorous poly(3‐hydroxybutrate‐co‐3‐hydroxyvalerate) (PHBV) film was prepared by solute leaching of salt/PHBV cast film. The surface chemistry of the PHBV membrane was modified by performing graft polymerization of methacrylic acid (MAA) on ozone treated porous PHBV film, followed by immobilization of type I collagen. The surface characteristics of the modified and nonmodified porous films were measured by water contact angle. The rat osteosarcoma cell line UMR‐106 osteoblast like cells were used as model cells to evaluate the cell viability on surfaces. The initial cell attachment, growth pattern, and proliferation as measured by MTT assay were used to evaluate the bone cell viability on the modified and nonmodified films. Among the PHBV films studied, the nonmodified porous PHBV and the porous PHBV film type I collagen dip coated showed no significant difference in cell attachment and proliferation, while the porous PHBV membrane that was collagen immobilized after MAA grafting showed considerable activity of osteoblast like cells. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 1916–1921, 2005

Poly(β-hydroxybutyrate-co-β-hydroxyvalerate) Supports in Vitro Osteogenesis

Studies have demonstrated that polymeric biomaterials have the potential to support osteoblast growth and development for bone tissue repair. Poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV), a bioabsorbable, biocompatible polyhydroxy acid polymer, is an excellent candidate that, as yet, has not been extensively investigated for this purpose. As such, we examined the attachment characteristics, self-renewal capacity, and osteogenic potential of osteoblast-like cells (MC3T3-E1 S14) when cultured on PHBV films compared with tissue culture polystyrene (TCP). Cells were assayed over 2 weeks and examined for changes in morphology, attachment, number and proliferation status, alkaline phosphatase (ALP) activity, calcium accumulation, nodule formation, and the expression of osteogenic genes. We found that these spindle-shaped MC3T3-E1 S14 cells made cell–cell and cell–substrate contact. Time-dependent cell attachment was shown to be accelerated on PHBV compared with collagen and laminin, but delayed compared with TCP and fibronectin. Cell number and the expression of ALP, osteopontin, and pro-collagen α1(I) mRNA were comparable for cells grown on PHBV and TCP, with all these markers increasing over time. This demonstrates the ability of PHBV to support osteoblast cell function. However, a lag was observed for cells on PHBV in comparison with those on TCP for proliferation, ALP activity, and cbfa-1 mRNA expression. In addition, we observed a reduction in total calcium accumulation, nodule formation, and osteocalcin mRNA expression. It is possible that this cellular response is a consequence of the contrasting surface properties of PHBV and TCP. The PHBV substrate used was rougher and more hydrophobic than TCP. Although further substrate analysis is required, we conclude that this polymer is a suitable candidate for the continued development as a biomaterial for bone tissue engineering.

Nanofabrication of microbial polyester by electrospinning promotes cell attachment

The biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as nanofibrous mats by electrospinning. Image analysis of the electrospun nanofibers fabricated from a 2 wt% 2,2,2-trifluoroethanol solution revealed a unimodal distribution pattern of fiber diameters with an observed average diameter of ca. 185 nm. The fiber diameter of electrospun fabrics could be controlled by adjusting the electrospinning parameters, including the solvent composition, concentration, applied voltage, and tip-to-collector distance. Chondrocytes derived from rabbit ear were cultured on a PHBV cast film and an electrospun PHBV nano-fibrous mat. After incubation for 2 h, the percentages of attached chondrocytes on the surfaces of the flat PHBV film and the PHBV nanofibrous mat were 19.0 and 30.1%, respectively. On the surface of the electrospun PHBV fabric, more chondrocytes were attached and appeared to have a much greater spreaded morphology than did that of the flat PHBV cast film in the early culture stage. The electrospun PHBV nanofabric provides an attractive structure for the attachment and growth of chondrocytes as cell culture surfaces for tissue engineering.

Bone cell viability on collagen immobilized poly(3‐hydroxybutrate‐co‐3‐hydroxyvalerate) membrane: Effect of surface chemistry

AbstractThe effect of surface chemistry on proliferation and morphology of bone cells cultured on surface modified poly(3‐hydroxybutrate‐co‐3‐hydroxyvalerate) (PHBV) and untreated PHBV was evaluated. The surface of cast PHBV film was physically and chemically immobilized with collagen. For preparing chemically immobilized collagen surface, PHBV film was ozone treated followed by grafting of PMAA chains and the immobilization of collagen. The surface roughness and hydrophilicity of PHBV film were determined by atomic force microscopy (AFM) and contact angle measurements, respectively. It was found that the duration of ozone exposure and monomer concentration used for grafting PMMA chains influenced the amount of collagen immobilized. The cell proliferation on PHBV surfaces with chemically and physically immobilized collagen was compared with untreated PHBV using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay. The bone cell activity on chemically and physically immobilized collagen PHBV films was found to be 246 and 107% for UMR‐106 and 68 and 9% for MC3T3 cell lines, respectively. Although the results are very preliminary, the chemically grafted collagen on PHBV surface provided a favorable matrix for cell proliferation. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2445–2453, 2004