BACKGROUND
advances in materials science and technology have led to rapid developments in regenerative medicine, tissue engineering and smart medical devices.
medical devices produced using synthetic polymers have been successfully applied in tissue engineering, serving as extracellular matrices to support cell growth, attachment and proliferation during in vitro cultivation and subsequent implantation  <cit> .
however, the response of tissues to implanted medical devices composed of synthetic polymers can cause several adverse effects; for example, cell adhesion on untreated polymers is insufficient, the hydrophobic polymer surface prevents cell in-growth, and the lack of functional residues complicates chemical modification.
furthermore, the acidic residues of products degraded in vivo often induce inflammation, which has negative effects on successful applications  <cit> .
the primary objective of tissue engineering is to reconstruct tissues and organs.
thus, implantable materials must provide mechanical support and must be compatible with cell behavior by promoting cell adhesion, proliferation and organization for the formation of functional tissue  <cit> .
over the past decade, polyhydroxyalkanoates , a family of polyesters produced by microorganisms that notably include poly , copolymers of poly , poly , copolymers of poly , copolymers of poly , poly  and their composites, have been widely applied to develop medical devices such as sutures, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings  <cit> .
by manipulating the copolymer compositions, a wide range of favorable mechanical properties and control over the degradation times under specific physiological conditions can be obtained.
various in vitro and in vivo tests have demonstrated that biomaterials composed of pha are compatible with bone, cartilage tissue, blood and various cell lines  <cit> .
moreover, the results of in vivo studies have demonstrated that these materials possess various degrees of biocompatibility and biodegradability with various cell lines, collagen and stem cells  <cit> .
thus, the properties of pha are being highlighted for applications in both conventional polymers and biomedical materials.
however, as thoroughly reviewed by chen and wu  <cit> , all studies concerning the biocompatibility of pha as a biomaterial involved evaluating the surface properties, which must satisfy requirements for the growth of different tissues and cells.
the fundamental objective of tissue engineering is to repair or replace damaged organs or tissues by delivering functional cells, supporting scaffolds, growth-promoting molecules  and electric or physiologic signals to areas in need  <cit> .
a number of studies have reported that biomaterials used for tissue engineering applications should be able to elicit specific responses from the cell and thereby direct cell attachment, proliferation, differentiation, and extracellular matrix production and organization  <cit> .
in particular, several studies involving the wound-healing process have reported that bioactiveinterleukin- <dig>  is expressed in wounds and enhances the wound-healing process.
both growth factors  and il-8) have important roles in enhancing the healing of wounds from burn injuries  <cit> .
the objective of this study was to evaluate the biocompatibilities of random and putative block phbvs produced by a model metabolic reaction-based system, which was successfully developed in a previous study  <cit> .
in the previous study, a model predictive control -based system consisting of two controller units was successfully developed.
the first mpc unit is for on-line monitoring and for controlling the concentrations of two types of alcohols, ethanol and n-pentanol, and the second mpc unit is the metabolic reaction  controller for automated control over the 3hv composition in phbv  <cit> .
the mr controller was constructed based on the metabolic flux distribution analysis of phbv biosynthesis in paracoccus denitrificans atcc  <dig>  <cit> .
because of the performance of this developed system, phbvs can be produced with arbitrary 3hv contents ranging from  <dig> to 90 mol%.
it was observed that the appearance of phbv films produced using the optimized method differed from that of phbv films produced using the conventional method.
next, the crystallization kinetics were evaluated under non-isothermal conditions using differential scanning calorimetry .
the results indicated that phbvs produced using the optimized method crystallized through a heterogeneous nucleation and three-dimensional growth process, whereas the phbvs produced using the conventional method crystallized through a one-dimensional growth process  <cit> .
this study focused on the biocompatibilities of random phbvs and of putative block phbvs produced by the model metabolic reaction-based system.
the copolymers obtained from different processing batches were fractionated.
the defined fractions were characterized based on their sequence distributions determined through 1h and 13c nmr analyses according to a previous report  <cit> .
the physical and mechanical properties of these two types of copolymers were also investigated using dsc and a universal testing machine.
to explore the potential of phas as biomaterials, the in vitro cytotoxicities of films composed of random phbvs and putative block phbvs toward l <dig> mouse connective tissue, human dermal fibroblast and saos- <dig> human osteosarcoma cells were investigated.
the biocompatibilities of the biomaterials were determined by monitoring the cell behavior during the adhesion of cells to their surfaces using sem and afm.
in this study, cell growth, adhesion, proliferation and morphology and mitochondrial function were analyzed.
additionally, quantitative analyses of tgf-β- <dig> and il- <dig> were conducted.
there have been no previous reports regarding the production of tgf-β- <dig> and il- <dig> by the cell lines being tested with pha.
to determine the usefulness of phb and phbv as biomaterials for the regeneration of tissue in problematic wounds due to the lack of il- <dig>  quantitative analyses of tgf-β- <dig> and il- <dig> are necessary.
based on the obtained results, the potential application of phb, random phbvs and putative block phbvs produced by a metabolic reaction-based system as biomaterials is discussed in terms of their properties.
RESULTS
1h and 13c nmr analyses of phbv films
phb, random phbvs and putative block phbvs were biosynthesized by p. denitrificans attc  <dig> using two different feeding methods, as mentioned above.
however, note that the phbvs produced in this study were obtained using different fed-batch cultivations, as reported previously  <cit> .
next, the produced phbvs were compositionally fractionated using a solution of chloroform and n-heptane, and the defined fractions were characterized using 1h and 13c nmr  <cit> .
this experiment focused on comparing the diad and triad sequence distributions between the random and putative block phbv samples.
the diad and triad sequence distributions were determined from the 600 mhz 13c nmr spectra, which are shown in figure  <dig>  the obtained relative peak intensities were interpreted in terms of the copolymer sequence distributions.
the expanded co signals of the 3hb unit  and of the 3hv unit  at  <dig> to 170 ppm were split into four peaks corresponding to diad sequences of b✳b, b✳v + v✳b, and v✳v.
it is clear that the v-centered triad sequences of the random phbv sample  had a predominantly random sequence character.
the diad and triad sequence distributions of the putative block phbv samples that were produced using the optimized method and that were well fractionated  did not show the multiple signals of b✳v + v✳b, and only the v✳v and b✳b signals were detected as a separated signal.
thus, the copolymer sequence distributions of the putative block phbv samples are different from those of the random phbvs.
it has been reported that the 13c nmr spectra of random and putative block phbv samples could exhibit a difference around 169–170 ppm and that they are sensitive to the influence of each carbonyl group of every component  <cit> .
next, the conditional probability for the copolymerization reaction of random phbv and putative phbv samples was determined by the nonlinear least squares method using the first-order markovian model.
it has been reported that this model can be applied to analyze random, block and alternative copolymerizations  <cit> .
the probability derived from the first-order markovian statistics and the d values of all samples are listed in table  <dig>  for the completely random phbv samples, the calculated d values are close to one.
for the putative block phbv samples, the calculated d values are considerably greater than one, and their observed 13c nmr spectra are distinctly different from those of typical random phbvs.figure  <dig>
the 600 mhz
13
c nmr spectra of the b <dig>  v <dig>  b <dig>  b <dig>  v <dig>  and v <dig> carbon resonances of  random phbv with 53 mol% of 3hv  and  putative block phbv with 50 mol% of 3hv .
the mechanical properties of films composed of phb, random and putative block-phbvs, commercialized phb, ps and pvc
sample
3hv mol%
d value
elongation  at max load
stress at max load
young’s modulus
toughness
random phbvs
putative block phbvs
mechanical properties
table  <dig> presents the copolymer compositions and mechanical properties of the phb homopolymer and of the random and putative block phbvs, which were prepared as film sheets and tested under the same conditions.
for comparison, commercial phb, pure ps and pvc were also tested under the same conditions.
note that none of the films produced in this study contain nucleating agents or plasticizers.
it was observed that the mechanical properties of the random phbvs differ from those of the putative block phbvs.
the highest values were observed for the phb, which exhibited a young’s modulus of <dig>  mpa and a stress at maximum load of  <dig>  mpa.
for the random phbvs consisting of 5%, 14%, 23%, 53%, 60%, 72% and 80% hv, the stress at maximum load and the young’s modulus decreased as the molar fraction of 3hv increased from  <dig> to 60 mol%, demonstrating that the random phbvs consisting of 0–60 mol% 3hv were more flexible and tougher.
the elongation required for breaking increased as the 3hv content increased from  <dig> to 23 mol%.
however, the random phbvs consisting of 72 mol% and 80 mol% hv became stiff and brittle, similar to the p. these results are consistent with those in the literature  <cit> .
notably, this is the first report on the mechanical properties of putative block phbvs consisting of 12% to 50% 3hv compared with random phbvs consisting of 5% to 72% 3hv produced by p. denitrificans.
the % elongation required for breaking and the toughness of the putative block phbvs increased as the 3hv content increased.
it was observed that the putative block phbvs consisting of 12% and 19% 3hv exhibited lower % elongation, toughness, young’s modulus, elongation and stress at maximum load than those of random phbvs containing similar 3hv contents.
however, the putative block phbvs with hv contents of 41% and 50% possessed higher % elongation, toughness, young’s modulus and stress at maximum load than those of random phbvs with similar 3hv contents.
compared with pure ps and pvc prepared using the same procedures, phb and random phb with 5% 3hv exhibited similar % elongations, but their toughness was lower than that of pvc.
the block phbv with 50% 3hv exhibited the highest toughness and % elongation among the polymers investigated in this study.
cytotoxicity evaluation
the cytotoxicities of pha film sheets composed of phb and of random and putative block phbvs were evaluated against three types of mammalian cell lines: l <dig> mouse connective tissue, saos- <dig> human osteosarcoma cells and human dermal fibroblasts.
the percent viabilities of the cells were compared to those of the cells cultured in 12-well plates and treated only with fresh medium .
the mtt conversion data are presented in figure  <dig> figure  <dig>
the cytotoxicity results of l <dig> mouse connective tissue, human dermal fibroblast and saos- <dig> human osteosarcoma cells when separately grown on films composed of phb, random and putative block phbvs, commercial phb, ps and pvc.
white bars, l <dig> mouse connective tissue; grey bars, saos- <dig> human osteosarcoma; black bars, human dermal fibroblast.
all three cell lines grew well on all of the produced pha film sheets.
in this study, the percent viability increased over time in all samples, including the control.
however, the l <dig> mouse connective tissue and human dermal fibroblasts clearly proliferated at significantly higher rates on the produced pha film sheets than on the chemically synthesized ps and pvc film sheets.
there was no significant difference between the random and putative block phbvs with respect to cell proliferation.
both appeared to be non-toxic to all three types of cell lines and exhibited percent viabilities that were better than those of the ps and pvc film sheets.
biocompatibility evaluation
to evaluate the biocompatibility for the attachment and proliferation of cells on the surfaces of the pha film sheets, each cell type was cultured on the surfaces of the films for 7 days.
the proliferation of the cells was determined every 24 hours for 7 days.
figure  <dig> presents the specific growth rates of l <dig> mouse connective tissue, saos- <dig> human osteosarcoma cells and human dermal fibroblasts on pha film sheets after the cells were cultured for 7 days.
the cell densities and cell line viabilities increased until confluency on day  <dig> for both l <dig> mouse connective tissue and saos- <dig> human osteosarcoma cells and on day  <dig> for dermal fibroblasts, whose cell density and viability subsequently declined until day  <dig>  it was observed that human dermal fibroblasts exhibited a specific growth rate that was slower than those of l <dig> mouse connective tissue and saos-2human osteosarcoma cells.
the effects of the monomer composition and the difference in the random and block sequence distributions on the specific growth rates of all three cell lines were not observed in this study.figure  <dig>
the specific growth rates of l <dig> mouse connective tissue, human dermal fibroblast and saos- <dig> human osteosarcoma cells when separately grown on films composed of phb, random and putative block phbvs, commercial phb, ps and pvc for 7 days.
white bars, l <dig> mouse connective tissue; grey bars, saos- <dig> human osteosarcoma; black bars, human dermal fibroblast.
production of tgf-β- <dig> and il-8
tgf-β- <dig> is a growth factor that is produced by various types of cells.
the amount of tgf-β- <dig> produced by cells is very low under normal conditions.
many studies have reported the involvement of tgf-β- <dig> and il- <dig> during wound healing, inflammation, remodeling and bone formation  <cit> .
in this study, l <dig> mouse connective tissue cells, human dermal fibroblast cells and saos- <dig> human osteosarcoma cells were cultured on the surfaces of the phb and random phbv and putative block phbv film sheets.
the amounts of tgf-β- <dig> and il- <dig> secreted into the cultured supernatant were determined using elisa test kits.
the control experiments were performed in the same way, except phb or phbv film sheets were not placed at the bottom of each well.
as shown in figure  <dig>  102– <dig>  1622– <dig> and 8483–9118 pg/mltgf-β- <dig> were produced from l <dig> mouse connective tissue , human dermal fibroblasts  and saos- <dig> human osteosarcoma cells , respectively, when grown on pha film sheets for 7 days.
however, there appears to be an obvious decrease in tgf-β- <dig> levels compared with their respective control levels, as shown in figures 4a,b and c. interestingly, opposite trends were observed when comparing the production of tgf-β- <dig> and il- <dig> from human dermal fibroblasts  and from saos- <dig> human osteosarcoma cells .
saos- <dig> osteosarcoma cells grown on pha film sheets produced more tgf-β- <dig> than il- <dig>  whereas human dermal fibroblasts grown on pha film sheets produced more il- <dig> than tgf-β- <dig>  notably, as shown in figure 4b, human dermal fibroblasts produced significantly more il- <dig> than did the controls, but they produced less tgf-β- <dig> than did the controls.
in figure 4a, the effect of the monomeric composition in phbv shows thatl <dig> mouse connective tissue cells grown on random 72%hv, random 80%hv, putative block 19%hv, putative block 41%hv and putative block 51%hv appear to have almost 2-fold higher levels of tgf-β- <dig> than l <dig> cells grown on phb and random phbv with low 3hvcontents.
the effect of the monomeric composition in putative block phbvs appears to be higher than that observed in random phbvs with similar 3hv contents.
however, the levels of tgf-β- <dig> produced from all three types of mammalian cells did not exceed the control levels.figure  <dig>
the production of tgf-β- <dig> and il- <dig> from  l <dig> mouse connective tissue,  human dermal fibroblast and  saos- <dig> human osteosarcoma cells when grown separately on films composed of phb and random and putative block phbvs for 7 days.
white bars, tgf-β-1; grey bars, il- <dig>
sem and afm analyses of the surfaces of phbvs with and without cell attachment
to assess the microstructures and morphologies of the film surfaces, sem and afm images of cells grown on phb and on random and block phbv film sheets were regularly recorded over one month.
figure  <dig> presents typical scanning electron micrographs  of the surface of a random phbv with 23% 3hv.
the original surface of the random phbv with 23% 3hv  was compared with the film surface after culturing human fibroblast cells on the film sheets for one month .
as shown in figure 5b, the human dermal fibroblast cells could grow and proliferate very well on the random phbvs.
the cells exhibited the normal and healthy characteristics of human dermal fibroblast cells.
figure 5c shows the opposite side of the same film sheet.
after culturing for one month, the film surface ultimately eroded; human dermal fibroblast cells were directly attached to the observed erosion sites .figure  <dig>
the sem images of human dermal fibroblasts cultured on a film sheet composed of random phbv with 23% 3hv content; the surface of the film sheet  before cell cultivation, bar  <dig> μm;  after cell cultivation for one month, bar  <dig> μm;  the opposite side of the same film sheet after cell cultivation for one month, bar  <dig> μm;  the same film with human dermal fibroblast cells grown on the surface for one month, bar  <dig> μm.
figure  <dig> presents the sems of surfaces of putative block phbvs with 19% 3hv under the same conditions.
figure 5a shows the original film sheet, and figures 6b,c and d show human dermal fibroblast cells and the surface of the film sheet after cell culturing for one month.
the human dermal fibroblasts grew very well on the putative block phbvs .
a small erosion of the film surface of putative block phbvs was also observed, but this erosion was less than that on the random phbvs.figure  <dig>
the sem images of human dermal fibroblasts cultured on a film sheet composed of putative block phbv with 19% 3hv content; the surface of the film sheet  before cell cultivation, bar  <dig> μm;  after cell cultivation for one month, bar  <dig> μm;  the opposite side of the same film sheet after cell cultivation for one month, bar  <dig> μm;  the same film with human dermal fibroblast cells grown on the surface for one month, bar  <dig> μm.
to evaluate these changes, tapping mode afm measurements were conducted using a scanning probe microscope, the nanoscope.
the results are shown in figures  <dig> and  <dig>  figure 7a and b show the original surface of random phbvs with 23% 3hv.
the film possesses a smooth surface.
however, after culturing human dermal fibroblast cells on the surfaces of the film sheets for one month, as shown in figure 7c and d, similar erosions of the film surface were observed as that in the sem analysis.
figure 8a and b show the original surface of block phbv with 19% 3hv.
the film surface of the block phbv was rich with crystalline regions and rougher than the random phbv surface with a similar 3hv content.
film surface erosion was not observed even after culturing human dermal fibroblast cells on the surface of film sheets for one month, as shown in figure 8c and d. the cells attached on the surface are shown in white.
similar observations were made for l <dig> mouse connective tissue and saos- <dig> human osteosarcoma cells.figure  <dig>
the afm images of human dermal fibroblasts cultured on a film sheet composed of random phbv with 23% 3hv content;  2-d image and  3-d image of the original surface of the film sheet before cell cultivation for one month;  2-d image and  3-d image of the same film sheet after cell cultivation for one month.
the afm images of human dermal fibroblasts cultured on a film sheet composed of putative block phbv with 19% 3hv content;  2-d image and  3-d image of the original surface of the film sheet before cell cultivation for one month;  2-d image and  3-d image of the same film sheet after cell cultivation for one month.
discussion
previous studies have shown that homopolymers of phb and random and putative block phbvs can be successfully produced with arbitrary 3hv contents from  <dig> to 80 mol% using an automated feed strategy based on a metabolic reaction system.
it was found that the different feeding strategies of mixed carbon sources, ethanol and n-pentanol, strongly affected the thermal properties of the phbv copolymer  <cit> , enzyme degradation rates  <cit>  and, in this study, mechanical properties.
the random and putative block phbvs exhibited different mechanical characteristics due to their different monomeric sequence distributions .
the enzyme degradation rates of random and putative block phbvs were different because the block phbv was rich in long-sequences of block 3hb units but less rich in 3hv random regions  <cit> .
the kinetics of the non-isothermal crystallization of block phbv was faster than that of the random phbv due to the frequent occurrence of block 3hb units  <cit> .
therefore, the difference in monomeric sequence distribution affects the thermal properties, biodegradable properties and mechanical properties  <cit> .
because the surface microstructure and monomeric sequence distribution may also affect the biocompatible properties, we investigated the biocompatibilities of the produced phb and random and putative block phbvs in vitro against three different mammalian cell lines.
first, the cytotoxicities of phb and random and putative block phbvs with 3hv contents from 0-80% were investigated using mtt assays .
the mtt assays and the cell growth kinetics revealed that phb and random and putative block phbvs showed evidence of non-toxicity toward l <dig> mouse connective tissue, human dermal fibroblast and saos-2human osteosarcoma cells, leading to increased proliferation in terms of increased cell viabilities and specific growth rates  <cit> .
however, decreases in cell viability and delays in cell growth were observed in ps and pvc.
this finding can be explained based on the fact that monomeric 3hba is an intermediate that naturally occurs in human blood  <cit> .
the surface properties of these phbv samples may promote cell attachment and proliferation by providing sufficient gas and nutrient exchange or more serum protein adsorption.
in addition, phbvs may possess adequate hydrophobic surface areas to induce the selective adsorption of cells because of the natural hydrophobic character of cells  <cit> .
in this study, the effects of the monomeric sequence distribution between random and block copolymers on cell viability and growth were not observed.
growth factors and cytokines are considered to be therapeutic candidates because they are synthesized by and stimulate cells required for tissue repair .
growth factors and cytokines are deficient in chronic wounds, and pharmacological applications on wounds enhance the wound repair process.
the wound-healing process normally occurs in three stages:  the directed and sequential migration of neutrophils, monocytes, keratinocytes and fibroblasts into the wound during the first several days;  the activation of wound macrophages and fibroblasts, resulting in the de novo synthesis of growth factors, cytokines and extracellular matrix proteins and the proliferation of fibroblasts in the successive 2– <dig> weeks; and  remodeling with active collagen turnover and cross linking from two weeks to one year  <cit> .
therefore, this study focused on the influences of phb and phbv on the production of tgf and il- <dig> by human dermal fibroblasts .
it was found that l <dig> mouse connective tissue and saos- <dig> human osteosarcoma cells, when grown on phb or random or putative block phbvs, did not produce significant amounts of tgf and il- <dig> compared with the control experiments.
however, human dermal fibroblasts secreted significant amounts of il- <dig> that were much higher than in the control experiment.
it is known that fibroblasts secrete a broad array of cytokines that are closely involved in the wound-healing process, including tgf-β- <dig> and il- <dig>  there has been little research regarding the effects of biomaterials on the production of tgf-β- <dig> and il- <dig> by human dermal fibroblast cells, except for the work by lv et al.
<cit> , who reported the systematic effects of chitosan on fibroblasts derived from hypertrophic scars and keloids.
three types of fibroblasts, hypertrophic scar fibroblasts, keloids fibroblasts and normal dermal fibroblasts, were cultured in the presence or absence of indicated dosages of chitosan, ranging from  <dig> to 320 μg/ml, for 72 hours.
these authors reported that treatment with chitosan could increase the production of il- <dig> but dramatically reduce the production of tgf-β- <dig>  both in a dose-dependent manner.
they concluded that chitosan regulated the production of tgf-β- <dig> and il- <dig>  providing more evidence that chitosan has the potential to be used as wound-healing and tissue repair agent or as a dressing material  <cit> .
in this study, phb and random and putative block phbvs may contribute factors that induce human dermal fibroblast cells to produce il- <dig>  with regards to the usefulness of phb and phbv as biomaterials for the regeneration of tissue, the production of il- <dig> which is induced by phb and phbvs, may be used to improve and enhance the wound-healing process because of deficiencies of il- <dig> in the wound area, particularly in problematic wounds such as burn infections and decubitus ulcer problems and in secondary wounds from systemic diseases.
the morphologic evaluation  revealed that human dermal fibroblast cells grew well on phb and random and block phbv matrices, which afm confirmed by revealing the three-dimensional growth of cells on the matrix.
combined with the results shown in figure  <dig>  the specific growth rates of l <dig> mouse connective tissue, saos- <dig> human osteosarcoma and human dermal fibroblast cells grown on ps were  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  1/h, respectively.
the specific growth rates of all three types of cell lines grown on standard phb, produced phb, random phbvs and putative block phbvs were higher than those on ps.
in contrast, pvc resulted in lower specific growth rates for all three types of cell lines compared to ps.
the produced phb and random and putative block phbvs were cell-supportive materials based on the appearance of spindle-shaped cells, which are attained only after proper cell adhesion to a substrate.
the sem and afm results clearly showed the ability of human dermal fibroblast cells to adhere and proliferate on random and block phbv surfaces.
at the same time, cell cultures of different origins, including l <dig> mouse connective tissue and saos- <dig> human osteosarcoma cells, in direct contact with phb and random and putative block phbvs films exhibited high levels of cell adhesion  <cit> .
indeed, the erosion rates of random and block phbv film surfaces were similar to those reported in a previous study performed using in vitro enzymatic degradation with pha depolymerase from p. lemoignei.
by comparison, the degradation rates of block phbv film sheets were slower than those of random phbv film sheets for similar ranges of mol% 3hv, indicating that the degradation rates of these two polyesters were dependent on the 3hv content and the monomeric sequence distribution  <cit> .
the results indicate that all of the pha films could be applied as biomaterials to support cell growth and cell attachment for all three cell lines.
finally, taken together with the cell proliferation, density and morphology of mammalian cells grown on these biodegradable film sheets, these results demonstrate the potential for using phas as biomaterials due to their ability to support mammalian cell growth.
CONCLUSIONS
the phb and the random and putative block phbvs produced by the fed-batch cultivation of p. denitrificans with a feeding strategy based on a model metabolic reaction-based system are biocompatible with three mammalian cell types and induce human dermal fibroblasts to secrete il- <dig> one of the cytokines required for the wound-healing process.
taken together, the results support the use of the random and putative block phbvs produced in this study as candidates for biomaterials in tissue engineering for connective tissue, bone and dermal fibroblast reconstruction.

 advances in materials science and technology have led to rapid developments in regenerative medicine, tissue engineering and smart medical devices. medical devices produced using synthetic polymers have been successfully applied in tissue engineering, serving as extracellular matrices to support cell growth, attachment and proliferation during in vitro cultivation and subsequent implantation. however, the response of tissues to implanted medical devices composed of synthetic polymers can cause several adverse effects; for example, cell adhesion on untreated polymers is insufficient, the hydrophobic polymer surface prevents cell in-growth, and the lack of functional residues complicates chemical modification. furthermore, the acidic residues of products degraded in vivo often induce inflammation, which has negative effects on successful applications. the primary objective of tissue engineering is to reconstruct tissues and organs. thus, implantable materials must provide mechanical support and must be compatible with cell behavior by promoting cell adhesion, proliferation and organization for the formation of functional tissue. over the past decade, polyhydroxyalkanoates, a family of polyesters produced by microorganisms that notably include poly, copolymers of poly, poly, copolymers of poly, copolymers of poly, poly and their composites, have been widely applied to develop medical devices such as sutures, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. by manipulating the copolymer compositions, a wide range of favorable mechanical properties and control over the degradation times under specific physiological conditions can be obtained. various in vitro and in vivo tests have demonstrated that biomaterials composed of pha are compatible with bone, cartilage tissue, blood and various cell lines. moreover, the results of in vivo studies have demonstrated that these materials possess various degrees of biocompatibility and biodegradability with various cell lines, collagen and stem cells. thus, the properties of pha are being highlighted for applications in both conventional polymers and biomedical materials. however, as thoroughly reviewed by chen and wu, all studies concerning the biocompatibility of pha as a biomaterial involved evaluating the surface properties, which must satisfy requirements for the growth of different tissues and cells. the fundamental objective of tissue engineering is to repair or replace damaged organs or tissues by delivering functional cells, supporting scaffolds, growth-promoting molecules and electric or physiologic signals to areas in need. a number of studies have reported that biomaterials used for tissue engineering applications should be able to elicit specific responses from the cell and thereby direct cell attachment, proliferation, differentiation, and extracellular matrix production and organization. in particular, several studies involving the wound-healing process have reported that bioactiveinterleukin- is expressed in wounds and enhances the wound-healing process. both growth factors and il-8) have important roles in enhancing the healing of wounds from burn injuries. the objective of this study was to evaluate the biocompatibilities of random and putative block phbvs produced by a model metabolic reaction-based system, which was successfully developed in a previous study. in the previous study, a model predictive control -based system consisting of two controller units was successfully developed. the first mpc unit is for on-line monitoring and for controlling the concentrations of two types of alcohols, ethanol and n-pentanol, and the second mpc unit is the metabolic reaction controller for automated control over the 3hv composition in phbv. the mr controller was constructed based on the metabolic flux distribution analysis of phbv biosynthesis in paracoccus denitrificans atcc. because of the performance of this developed system, phbvs can be produced with arbitrary 3hv contents ranging from to 90 mol%. it was observed that the appearance of phbv films produced using the optimized method differed from that of phbv films produced using the conventional method. next, the crystallization kinetics were evaluated under non-isothermal conditions using differential scanning calorimetry. the results indicated that phbvs produced using the optimized method crystallized through a heterogeneous nucleation and three-dimensional growth process, whereas the phbvs produced using the conventional method crystallized through a one-dimensional growth process. this study focused on the biocompatibilities of random phbvs and of putative block phbvs produced by the model metabolic reaction-based system. the copolymers obtained from different processing batches were fractionated. the defined fractions were characterized based on their sequence distributions determined through 1h and 13c nmr analyses according to a previous report. the physical and mechanical properties of these two types of copolymers were also investigated using dsc and a universal testing machine. to explore the potential of phas as biomaterials, the in vitro cytotoxicities of films composed of random phbvs and putative block phbvs toward l mouse connective tissue, human dermal fibroblast and saos- human osteosarcoma cells were investigated. the biocompatibilities of the biomaterials were determined by monitoring the cell behavior during the adhesion of cells to their surfaces using sem and afm. in this study, cell growth, adhesion, proliferation and morphology and mitochondrial function were analyzed. additionally, quantitative analyses of tgf-β- and il- were conducted. there have been no previous reports regarding the production of tgf-β- and il- by the cell lines being tested with pha. to determine the usefulness of phb and phbv as biomaterials for the regeneration of tissue in problematic wounds due to the lack of il- quantitative analyses of tgf-β- and il- are necessary. based on the obtained results, the potential application of phb, random phbvs and putative block phbvs produced by a metabolic reaction-based system as biomaterials is discussed in terms of their properties.  1h and 13c nmr analyses of phbv films phb, random phbvs and putative block phbvs were biosynthesized by p. denitrificans attc using two different feeding methods, as mentioned above. however, note that the phbvs produced in this study were obtained using different fed-batch cultivations, as reported previously. next, the produced phbvs were compositionally fractionated using a solution of chloroform and n-heptane, and the defined fractions were characterized using 1h and 13c nmr. this experiment focused on comparing the diad and triad sequence distributions between the random and putative block phbv samples. the diad and triad sequence distributions were determined from the 600 mhz 13c nmr spectra, which are shown in figure  the obtained relative peak intensities were interpreted in terms of the copolymer sequence distributions. the expanded co signals of the 3hb unit and of the 3hv unit at to 170 ppm were split into four peaks corresponding to diad sequences of b✳b, b✳v + v✳b, and v✳v. it is clear that the v-centered triad sequences of the random phbv sample had a predominantly random sequence character. the diad and triad sequence distributions of the putative block phbv samples that were produced using the optimized method and that were well fractionated did not show the multiple signals of b✳v + v✳b, and only the v✳v and b✳b signals were detected as a separated signal. thus, the copolymer sequence distributions of the putative block phbv samples are different from those of the random phbvs. it has been reported that the 13c nmr spectra of random and putative block phbv samples could exhibit a difference around 169–170 ppm and that they are sensitive to the influence of each carbonyl group of every component. next, the conditional probability for the copolymerization reaction of random phbv and putative phbv samples was determined by the nonlinear least squares method using the first-order markovian model. it has been reported that this model can be applied to analyze random, block and alternative copolymerizations. the probability derived from the first-order markovian statistics and the d values of all samples are listed in table  for the completely random phbv samples, the calculated d values are close to one. for the putative block phbv samples, the calculated d values are considerably greater than one, and their observed 13c nmr spectra are distinctly different from those of typical random phbvs.figure the 600 mhz 13 c nmr spectra of the b v b b v and v carbon resonances of random phbv with 53 mol% of 3hv and putative block phbv with 50 mol% of 3hv. the mechanical properties of films composed of phb, random and putative block-phbvs, commercialized phb, ps and pvc sample 3hv mol% d value elongation at max load stress at max load young’s modulus toughness random phbvs putative block phbvs mechanical properties table  presents the copolymer compositions and mechanical properties of the phb homopolymer and of the random and putative block phbvs, which were prepared as film sheets and tested under the same conditions. for comparison, commercial phb, pure ps and pvc were also tested under the same conditions. note that none of the films produced in this study contain nucleating agents or plasticizers. it was observed that the mechanical properties of the random phbvs differ from those of the putative block phbvs. the highest values were observed for the phb, which exhibited a young’s modulus of  mpa and a stress at maximum load of  mpa. for the random phbvs consisting of 5%, 14%, 23%, 53%, 60%, 72% and 80% hv, the stress at maximum load and the young’s modulus decreased as the molar fraction of 3hv increased from to 60 mol%, demonstrating that the random phbvs consisting of 0–60 mol% 3hv were more flexible and tougher. the elongation required for breaking increased as the 3hv content increased from to 23 mol%. however, the random phbvs consisting of 72 mol% and 80 mol% hv became stiff and brittle, similar to the p. these results are consistent with those in the literature. notably, this is the first report on the mechanical properties of putative block phbvs consisting of 12% to 50% 3hv compared with random phbvs consisting of 5% to 72% 3hv produced by p. denitrificans. the % elongation required for breaking and the toughness of the putative block phbvs increased as the 3hv content increased. it was observed that the putative block phbvs consisting of 12% and 19% 3hv exhibited lower % elongation, toughness, young’s modulus, elongation and stress at maximum load than those of random phbvs containing similar 3hv contents. however, the putative block phbvs with hv contents of 41% and 50% possessed higher % elongation, toughness, young’s modulus and stress at maximum load than those of random phbvs with similar 3hv contents. compared with pure ps and pvc prepared using the same procedures, phb and random phb with 5% 3hv exhibited similar % elongations, but their toughness was lower than that of pvc. the block phbv with 50% 3hv exhibited the highest toughness and % elongation among the polymers investigated in this study. cytotoxicity evaluation the cytotoxicities of pha film sheets composed of phb and of random and putative block phbvs were evaluated against three types of mammalian cell lines: l mouse connective tissue, saos- human osteosarcoma cells and human dermal fibroblasts. the percent viabilities of the cells were compared to those of the cells cultured in 12-well plates and treated only with fresh medium. the mtt conversion data are presented in figure  figure the cytotoxicity results of l mouse connective tissue, human dermal fibroblast and saos- human osteosarcoma cells when separately grown on films composed of phb, random and putative block phbvs, commercial phb, ps and pvc. white bars, l mouse connective tissue; grey bars, saos- human osteosarcoma; black bars, human dermal fibroblast. all three cell lines grew well on all of the produced pha film sheets. in this study, the percent viability increased over time in all samples, including the control. however, the l mouse connective tissue and human dermal fibroblasts clearly proliferated at significantly higher rates on the produced pha film sheets than on the chemically synthesized ps and pvc film sheets. there was no significant difference between the random and putative block phbvs with respect to cell proliferation. both appeared to be non-toxic to all three types of cell lines and exhibited percent viabilities that were better than those of the ps and pvc film sheets. biocompatibility evaluation to evaluate the biocompatibility for the attachment and proliferation of cells on the surfaces of the pha film sheets, each cell type was cultured on the surfaces of the films for 7 days. the proliferation of the cells was determined every 24 hours for 7 days. figure  presents the specific growth rates of l mouse connective tissue, saos- human osteosarcoma cells and human dermal fibroblasts on pha film sheets after the cells were cultured for 7 days. the cell densities and cell line viabilities increased until confluency on day for both l mouse connective tissue and saos- human osteosarcoma cells and on day for dermal fibroblasts, whose cell density and viability subsequently declined until day it was observed that human dermal fibroblasts exhibited a specific growth rate that was slower than those of l mouse connective tissue and saos-2human osteosarcoma cells. the effects of the monomer composition and the difference in the random and block sequence distributions on the specific growth rates of all three cell lines were not observed in this study.figure the specific growth rates of l mouse connective tissue, human dermal fibroblast and saos- human osteosarcoma cells when separately grown on films composed of phb, random and putative block phbvs, commercial phb, ps and pvc for 7 days. white bars, l mouse connective tissue; grey bars, saos- human osteosarcoma; black bars, human dermal fibroblast. production of tgf-β- and il-8 tgf-β- is a growth factor that is produced by various types of cells. the amount of tgf-β- produced by cells is very low under normal conditions. many studies have reported the involvement of tgf-β- and il- during wound healing, inflammation, remodeling and bone formation. in this study, l mouse connective tissue cells, human dermal fibroblast cells and saos- human osteosarcoma cells were cultured on the surfaces of the phb and random phbv and putative block phbv film sheets. the amounts of tgf-β- and il- secreted into the cultured supernatant were determined using elisa test kits. the control experiments were performed in the same way, except phb or phbv film sheets were not placed at the bottom of each well. as shown in figure  102– 1622– and 8483–9118 pg/mltgf-β- were produced from l mouse connective tissue, human dermal fibroblasts and saos- human osteosarcoma cells, respectively, when grown on pha film sheets for 7 days. however, there appears to be an obvious decrease in tgf-β- levels compared with their respective control levels, as shown in figures 4a,b and c. interestingly, opposite trends were observed when comparing the production of tgf-β- and il- from human dermal fibroblasts and from saos- human osteosarcoma cells. saos- osteosarcoma cells grown on pha film sheets produced more tgf-β- than il- whereas human dermal fibroblasts grown on pha film sheets produced more il- than tgf-β- notably, as shown in figure 4b, human dermal fibroblasts produced significantly more il- than did the controls, but they produced less tgf-β- than did the controls. in figure 4a, the effect of the monomeric composition in phbv shows thatl mouse connective tissue cells grown on random 72%hv, random 80%hv, putative block 19%hv, putative block 41%hv and putative block 51%hv appear to have almost 2-fold higher levels of tgf-β- than l cells grown on phb and random phbv with low 3hvcontents. the effect of the monomeric composition in putative block phbvs appears to be higher than that observed in random phbvs with similar 3hv contents. however, the levels of tgf-β- produced from all three types of mammalian cells did not exceed the control levels.figure the production of tgf-β- and il- from l mouse connective tissue, human dermal fibroblast and saos- human osteosarcoma cells when grown separately on films composed of phb and random and putative block phbvs for 7 days. white bars, tgf-β-1; grey bars, il- sem and afm analyses of the surfaces of phbvs with and without cell attachment to assess the microstructures and morphologies of the film surfaces, sem and afm images of cells grown on phb and on random and block phbv film sheets were regularly recorded over one month. figure  presents typical scanning electron micrographs of the surface of a random phbv with 23% 3hv. the original surface of the random phbv with 23% 3hv was compared with the film surface after culturing human fibroblast cells on the film sheets for one month. as shown in figure 5b, the human dermal fibroblast cells could grow and proliferate very well on the random phbvs. the cells exhibited the normal and healthy characteristics of human dermal fibroblast cells. figure 5c shows the opposite side of the same film sheet. after culturing for one month, the film surface ultimately eroded; human dermal fibroblast cells were directly attached to the observed erosion sites.figure the sem images of human dermal fibroblasts cultured on a film sheet composed of random phbv with 23% 3hv content; the surface of the film sheet before cell cultivation, bar μm; after cell cultivation for one month, bar μm; the opposite side of the same film sheet after cell cultivation for one month, bar μm; the same film with human dermal fibroblast cells grown on the surface for one month, bar μm. figure  presents the sems of surfaces of putative block phbvs with 19% 3hv under the same conditions. figure 5a shows the original film sheet, and figures 6b,c and d show human dermal fibroblast cells and the surface of the film sheet after cell culturing for one month. the human dermal fibroblasts grew very well on the putative block phbvs. a small erosion of the film surface of putative block phbvs was also observed, but this erosion was less than that on the random phbvs.figure the sem images of human dermal fibroblasts cultured on a film sheet composed of putative block phbv with 19% 3hv content; the surface of the film sheet before cell cultivation, bar μm; after cell cultivation for one month, bar μm; the opposite side of the same film sheet after cell cultivation for one month, bar μm; the same film with human dermal fibroblast cells grown on the surface for one month, bar μm. to evaluate these changes, tapping mode afm measurements were conducted using a scanning probe microscope, the nanoscope. the results are shown in figures  and figure 7a and b show the original surface of random phbvs with 23% 3hv. the film possesses a smooth surface. however, after culturing human dermal fibroblast cells on the surfaces of the film sheets for one month, as shown in figure 7c and d, similar erosions of the film surface were observed as that in the sem analysis. figure 8a and b show the original surface of block phbv with 19% 3hv. the film surface of the block phbv was rich with crystalline regions and rougher than the random phbv surface with a similar 3hv content. film surface erosion was not observed even after culturing human dermal fibroblast cells on the surface of film sheets for one month, as shown in figure 8c and d. the cells attached on the surface are shown in white. similar observations were made for l mouse connective tissue and saos- human osteosarcoma cells.figure the afm images of human dermal fibroblasts cultured on a film sheet composed of random phbv with 23% 3hv content; 2-d image and 3-d image of the original surface of the film sheet before cell cultivation for one month; 2-d image and 3-d image of the same film sheet after cell cultivation for one month. the afm images of human dermal fibroblasts cultured on a film sheet composed of putative block phbv with 19% 3hv content; 2-d image and 3-d image of the original surface of the film sheet before cell cultivation for one month; 2-d image and 3-d image of the same film sheet after cell cultivation for one month. discussion previous studies have shown that homopolymers of phb and random and putative block phbvs can be successfully produced with arbitrary 3hv contents from to 80 mol% using an automated feed strategy based on a metabolic reaction system. it was found that the different feeding strategies of mixed carbon sources, ethanol and n-pentanol, strongly affected the thermal properties of the phbv copolymer, enzyme degradation rates and, in this study, mechanical properties. the random and putative block phbvs exhibited different mechanical characteristics due to their different monomeric sequence distributions. the enzyme degradation rates of random and putative block phbvs were different because the block phbv was rich in long-sequences of block 3hb units but less rich in 3hv random regions. the kinetics of the non-isothermal crystallization of block phbv was faster than that of the random phbv due to the frequent occurrence of block 3hb units. therefore, the difference in monomeric sequence distribution affects the thermal properties, biodegradable properties and mechanical properties. because the surface microstructure and monomeric sequence distribution may also affect the biocompatible properties, we investigated the biocompatibilities of the produced phb and random and putative block phbvs in vitro against three different mammalian cell lines. first, the cytotoxicities of phb and random and putative block phbvs with 3hv contents from 0-80% were investigated using mtt assays. the mtt assays and the cell growth kinetics revealed that phb and random and putative block phbvs showed evidence of non-toxicity toward l mouse connective tissue, human dermal fibroblast and saos-2human osteosarcoma cells, leading to increased proliferation in terms of increased cell viabilities and specific growth rates. however, decreases in cell viability and delays in cell growth were observed in ps and pvc. this finding can be explained based on the fact that monomeric 3hba is an intermediate that naturally occurs in human blood. the surface properties of these phbv samples may promote cell attachment and proliferation by providing sufficient gas and nutrient exchange or more serum protein adsorption. in addition, phbvs may possess adequate hydrophobic surface areas to induce the selective adsorption of cells because of the natural hydrophobic character of cells. in this study, the effects of the monomeric sequence distribution between random and block copolymers on cell viability and growth were not observed. growth factors and cytokines are considered to be therapeutic candidates because they are synthesized by and stimulate cells required for tissue repair. growth factors and cytokines are deficient in chronic wounds, and pharmacological applications on wounds enhance the wound repair process. the wound-healing process normally occurs in three stages: the directed and sequential migration of neutrophils, monocytes, keratinocytes and fibroblasts into the wound during the first several days; the activation of wound macrophages and fibroblasts, resulting in the de novo synthesis of growth factors, cytokines and extracellular matrix proteins and the proliferation of fibroblasts in the successive 2– weeks; and remodeling with active collagen turnover and cross linking from two weeks to one year. therefore, this study focused on the influences of phb and phbv on the production of tgf and il- by human dermal fibroblasts. it was found that l mouse connective tissue and saos- human osteosarcoma cells, when grown on phb or random or putative block phbvs, did not produce significant amounts of tgf and il- compared with the control experiments. however, human dermal fibroblasts secreted significant amounts of il- that were much higher than in the control experiment. it is known that fibroblasts secrete a broad array of cytokines that are closely involved in the wound-healing process, including tgf-β- and il- there has been little research regarding the effects of biomaterials on the production of tgf-β- and il- by human dermal fibroblast cells, except for the work by lv et al., who reported the systematic effects of chitosan on fibroblasts derived from hypertrophic scars and keloids. three types of fibroblasts, hypertrophic scar fibroblasts, keloids fibroblasts and normal dermal fibroblasts, were cultured in the presence or absence of indicated dosages of chitosan, ranging from to 320 μg/ml, for 72 hours. these authors reported that treatment with chitosan could increase the production of il- but dramatically reduce the production of tgf-β- both in a dose-dependent manner. they concluded that chitosan regulated the production of tgf-β- and il- providing more evidence that chitosan has the potential to be used as wound-healing and tissue repair agent or as a dressing material. in this study, phb and random and putative block phbvs may contribute factors that induce human dermal fibroblast cells to produce il- with regards to the usefulness of phb and phbv as biomaterials for the regeneration of tissue, the production of il- which is induced by phb and phbvs, may be used to improve and enhance the wound-healing process because of deficiencies of il- in the wound area, particularly in problematic wounds such as burn infections and decubitus ulcer problems and in secondary wounds from systemic diseases. the morphologic evaluation revealed that human dermal fibroblast cells grew well on phb and random and block phbv matrices, which afm confirmed by revealing the three-dimensional growth of cells on the matrix. combined with the results shown in figure  the specific growth rates of l mouse connective tissue, saos- human osteosarcoma and human dermal fibroblast cells grown on ps were  ± ,  ±  and  ±  1/h, respectively. the specific growth rates of all three types of cell lines grown on standard phb, produced phb, random phbvs and putative block phbvs were higher than those on ps. in contrast, pvc resulted in lower specific growth rates for all three types of cell lines compared to ps. the produced phb and random and putative block phbvs were cell-supportive materials based on the appearance of spindle-shaped cells, which are attained only after proper cell adhesion to a substrate. the sem and afm results clearly showed the ability of human dermal fibroblast cells to adhere and proliferate on random and block phbv surfaces. at the same time, cell cultures of different origins, including l mouse connective tissue and saos- human osteosarcoma cells, in direct contact with phb and random and putative block phbvs films exhibited high levels of cell adhesion. indeed, the erosion rates of random and block phbv film surfaces were similar to those reported in a previous study performed using in vitro enzymatic degradation with pha depolymerase from p. lemoignei. by comparison, the degradation rates of block phbv film sheets were slower than those of random phbv film sheets for similar ranges of mol% 3hv, indicating that the degradation rates of these two polyesters were dependent on the 3hv content and the monomeric sequence distribution. the results indicate that all of the pha films could be applied as biomaterials to support cell growth and cell attachment for all three cell lines. finally, taken together with the cell proliferation, density and morphology of mammalian cells grown on these biodegradable film sheets, these results demonstrate the potential for using phas as biomaterials due to their ability to support mammalian cell growth.  the phb and the random and putative block phbvs produced by the fed-batch cultivation of p. denitrificans with a feeding strategy based on a model metabolic reaction-based system are biocompatible with three mammalian cell types and induce human dermal fibroblasts to secrete il- one of the cytokines required for the wound-healing process. taken together, the results support the use of the random and putative block phbvs produced in this study as candidates for biomaterials in tissue engineering for connective tissue, bone and dermal fibroblast reconstruction. 
the produced phbvs were fractionated, and the copolymer sequence distributions were analyzed using 1h and 13c nmr spectroscopy. the diad and triad sequence distributions indicated that the phbvs produced via the fed-batch cultivation using two different feed systems resulted in two types of copolymers: random phbvs and putative block phbvs. the monomer compositions and sequence distributions strongly affected the thermal and mechanical properties. the in vitro cytotoxicities of films composed of random phbvs and putative block phbvs were investigated against three types of mammalian cells. films composed of poly, random phbvs, putative block phbvs, polystyrene and polyvinylchloride were prepared and characterized. notably, the random phbvs possessed different mechanical properties than the putative block phbvs. taken together, the results support the use of phb and the random and putative block phbvs produced in this study as potential biomaterials in tissue engineering applications for connective tissue, bone and dermal fibroblast reconstruction. the biocompatibilities of these films were evaluated in vitro against three types of mammalian cells: l mouse connective tissue, human dermal fibroblast and saos- human osteosarcoma cells. the mechanical integrity and characteristics of the film surfaces changed with the hv content. erosion of the pha film surfaces was observed by scanning electron microscopy and atomic force microscopy. none of the phbv films exhibited cytotoxic responses to the three types of mammalian cells. the production of il- which is induced by phb and phbvs, may be used to improve and enhance the wound-healing process because of deficiencies of il- in the wound area, particularly in problematic wounds. the thermal properties were analyzed using differential scanning calorimetry.
 this study evaluated the biocompatibilities of random and putative block polys produced by a metabolic reaction-based system. the produced phbvs were fractionated, and the copolymer sequence distributions were analyzed using 1h and 13c nmr spectroscopy. the thermal properties were analyzed using differential scanning calorimetry. mechanical tests were conducted using a universal testing machine. the in vitro cytotoxicities of films composed of random phbvs and putative block phbvs were investigated against three types of mammalian cells. the surfaces of the copolymer films and the morphologies of the cells were qualitatively monitored using scanning electron microscopy and atomic force microscopy.  films composed of poly, random phbvs, putative block phbvs, polystyrene and polyvinylchloride were prepared and characterized. the diad and triad sequence distributions indicated that the phbvs produced via the fed-batch cultivation using two different feed systems resulted in two types of copolymers: random phbvs and putative block phbvs. the monomer compositions and sequence distributions strongly affected the thermal and mechanical properties. the mechanical integrity and characteristics of the film surfaces changed with the hv content. notably, the random phbvs possessed different mechanical properties than the putative block phbvs. the biocompatibilities of these films were evaluated in vitro against three types of mammalian cells: l mouse connective tissue, human dermal fibroblast and saos- human osteosarcoma cells. none of the phbv films exhibited cytotoxic responses to the three types of mammalian cells. erosion of the pha film surfaces was observed by scanning electron microscopy and atomic force microscopy. the production of transforming growth factor-β- and interleukin- was also examined with regards to the usefulness of phb and phbv as biomaterials for regenerative tissue. the production of il- which is induced by phb and phbvs, may be used to improve and enhance the wound-healing process because of deficiencies of il- in the wound area, particularly in problematic wounds.  taken together, the results support the use of phb and the random and putative block phbvs produced in this study as potential biomaterials in tissue engineering applications for connective tissue, bone and dermal fibroblast reconstruction. keywords biocompatibilitybiodegradable polymerpolyhydroxyalkanoatesrandom copolymerblock copolymerphbvbiodegradationcytokinescytotoxicityissue-copyright-statement© the author 2014 