This study was designed to investigate the surface properties especially surface porosity of polyhydroxybutyrate (PHB) using scanning electron microscopy. PHB granules were sprinkled on the double-sided sticky tape attached on a SEM aluminium stub and sputtered with gold(10nm thickness) in a Polaron SC515 Coater, following which the samples were placed into the SEM specimen chamber for viewing and recording. Scanning electron micrographs with different magnification of PHB surface revealed multiple pores with different sizes.
This work aims to shed light in the fabrication of poly(3-hydroxybutyrate-co-44%-4-hydroxybutyrate)[P(3HB-co-44%4HB)]/chitosan-based silver nanocomposite material using different contents of silver nanoparticle (SNP); 1-9 wt%. Two approaches were applied in the fabrication; namely solvent casting and chemical crosslinking via glutaraldehyde (GA). A detailed characterization was conducted in order to yield information regarding the nanocomposite material. X-ray diffraction analysis exhibited the nature of the three components that exist in the nanocomposite films: P(3HB-co-4HB), chitosan, and SNP. In term of mechanical properties, tensile strength, and elongation at break were significantly improved up to 125% and 22%, respectively with the impregnation of the SNP. The melting temperature of the nanocomposite materials was increased whereas their thermal stability was slightly changed. Scanning electron microscopy images revealed that incorporation of 9 wt% of SNP caused agglomeration but the surface roughness of the material was significantly improved with the loading. Staphylococcus aureus and Escherichia coli were completely inhibited by the nanocomposite films with 7 and 9 wt% of SNP, respectively. On the other hand, degradation of the nanocomposite materials outweighed the degradation of the pure copolymer. These bioactive and biodegradable materials stand a good chance to serve the vast need of biomedical applications namely management and care of wound as wound dressing.
Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.
The main focus of this study is the incorporation of collagen peptides to fabricate P(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] nano-fiber construct to further enhance surface wettability and support cell growth while harbouring desired properties for biodegradable wound dressing. Simultaneous electrospinning of nanofiber P(3HB-co-4HB)/collagen peptides construct was carried out using dual syringe system. The wettability of the constructs increased with the increase in 4HB molar fraction from 20mol% 4HB [53.2°], P(3HB-co-35mol%4HB)[48.9°], P(3HB-co-50mol%4HB)[44.5°] and P(3HB-co-82mol%4HB) [37.7°]. In vitro study carried out using mouse fibroblast cells (L929) grown on nanofiber P(3HB-co-4HB)/collagen peptides construct showed an increase in cell proliferation. In vivo study using animal model (Sprague Dawley rats) showed that nanofibrous P(3HB-co-4HB)/collagen peptides construct had a significant effect on wound contractions with the highest percentage of wound closure of 79%. Hence, P(3HB-co-4HB)/collagen peptides construct suitable for wound dressing have been developed using nano-fabrication technique.
P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74-92 wt% and 4HB monomer composition of 92-99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).
Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M (n)) of copolymers ranged from 260 x 10(3) to 590 x 10(3)Da, and the polydispersities (M (w)/M (n)) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T (m)), glass transition temperature (T (g)), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.
Two-stage fermentation was normally employed to achieve a high poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] productivity with higher 4HB molar fraction. Here, we demonstrated single-stage fermentation method which is more industrial feasible by implementing mixed-substrate cultivation strategy. Studies on bioreactor scale show a remarkably high PHA accumulation of 73 wt%, contributing to a high PHA concentration and product yield of 8.6 g/L and 2.7 g/g, respectively. This fermentation strategy has resulted in copolymers with wider range of 4HB monomer composition, which ranges from 12 to 55 mol%. These copolymers show a broad range of weight average molecular weight (M w ) from 119.5 to 407.0 kDa. The copolymer characteristics were found to be predominantly affected by the nature of the substrates and the mixture strategies, regardless of the 4HB monomer compositions. This was supported by the determination of copolymer randomness using (13)C-NMR analysis. The study warrants significantly in the copolymer scale-up and modeling at industrial level.
The sustainability in polyhydroxyalkanoates (PHA) production is drawing increasing attention as the effort to increase the economic feasibility for commercialization pursues. Oleic acid is widely preferred by bacteria but its employment for PHA production makes sustainability rather dubious. This study showed promising poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] content of 68 wt % by lipase genes-harbouring Cupriavidus malaysiensis USMAA2-4 transformant from palm olein and 1-pentanol. High oleic acid content and low oil saturation caused palm olein to outperform crude palm oil, crude palm kernel oil and soybean oil due to its preference for oleic acid shown by previous screening. The transformant showed 8-fold and 40-fold higher lipase activity compared to C. necator H16 and its wild-type respectively. The transformant was unaffected by Co2+ but the growth of C. necator H16 was inversely proportional to Co2+ concentration and the employment of 1-pentanol also ceased its growth and PHA accumulation. Although the inhibitory effect of Fe2+, Cu2+ and Zn2+ at high molarity on LipA decreased PHA content of C. malaysiensis USMAA2-4 transformant by 23-24 wt %, the lipase activity was restorable with high molarity of Ca2+, thus resulted in higher PHA content. The transformant enabled the employment of low-cost 1-pentanol as the precursor for cost-effective PHA production and its preference for palm olein contributed to higher sustainability.
Polyhydroxyalkanoate (PHA) is a microbial polymer that has been at the forefront of many attempts at tissue engineering. However, the surface of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) is hydrophobic with few recognition sites for cell attachment. Various concentrations of fish-scale collagen peptides (FSCPs) were incorporated into P(3HB-co-4HB) copolymer by aminolysis. Later, FSCPs were introduced onto the aminolyzed P(3HB-co-4HB) scaffolds. Introduction of the FSCP groups was verified using Fourier transform infrared spectroscopy and the ninhydrin method. The effect of the incorporation of FSCPs on hydrophilicity was investigated using the water contact angle. As the concentration of FSCPs increased, the water contact angle decreased. In vitro study demonstrated that P(3HB-co-4HB)/FSCP scaffolds provided better cell attachment and growth of L929 mouse fibroblast cells and better cell proliferation. In vivo study showed that P(3HB-co-4HB)/1.5 wt% FSCPs had a significant effect on wound contractions, with the highest percentage of wound closure (61%) in 7 d.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [(P(3HB-co-4HB)] copolymer receives attention as next generation biomaterial in medical application. However, the exploitation of the copolymer is still constrained since such copolymer has not yet successfully been performed in industrial scale production. In this work, we intended to establish pilot production system of the copolymer retaining the copolymer quality which has recently discovered to have novel characteristic from lab scale fermentation. An increase of agitation speed has significantly improved the copolymer accumulation efficiency by minimizing the utilization of substrates towards cell growth components. This is evidenced by a drastic increase of PHA content from 28 wt% to 63 wt% and PHA concentration from 3.1 g/L to 6.5 g/L but accompanied by the reduction of residual biomass from 8.0 g/L to 3.8 g/L. Besides, fermentations at lower agitation and aeration have resulted in reduced molecular weight and mechanical strength of the copolymer, suggesting the role of sufficient oxygen supply efficiency in improving the properties of the resulting copolymers. The KLa-based scale-up fermentation was performed successfully in maintaining the yield and the quality of the copolymers produced without a drastic fluctuation. This suggests that the scale-up based on the KLa values supported the fermentation system of P(3HB-co-4HB) copolymer production in single-stage using mixed-substrate cultivation strategy.
This study reports the production of P(3HB-co-4HB) [Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)] in possession of high molecular weight and elastomeric properties by Cupriavidus sp. USMAA1020 in single-stage mixed-substrate cultivation system. 1,4-butanediol and 1,6-hexanediol are found to be efficient substrate mixture that has resulted in high copolymer yield, occupying a maximum of 70wt% of the total biomass and producing higher 4HB monomer composition ranging from 31mol% to 41mol%. In substrate mixtures involving 1,6-hexanediol, cleavage of the 6-hydroxyhexanoyl-CoA produces Acetyl-CoA and 4-hydroxybutyryl-CoA. Acetyl-CoA is instrumental in initiating the cell growth in the single-stage fermentation system, preventing 4-hydroxybutyryl-CoA from being utilized via β-oxidation and retained the 4HB monomer at higher ratios. Macroscopic kinetic models of the bioprocesses have revealed that the P(3HB-co-4HB) formation appears to be in the nature of mixed-growth associated with higher formation rate during exponential growth phase; evidenced by higher growth associated constants, α, from 0.0690g/g to 0.4615g/g compared to non-growth associated constants, β, from 0.0092g/g/h to 0.0459g/g/h. The P(3HB-co-31mol% 4HB) produced from the substrate mixture exhibited high weight-average molecular weight, Mwof 927kDa approaching a million Dalton, and possessed elongation at break of 1637% upon cultivation at 0.56wt% C. This is the first report on such properties for the P(3HB-co-4HB) copolymer. The copolymer is highly resistant to polymer deformation after being stretched.
Long carbon chain alkanediols are used in the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], however these substrates possess high toxicity towards bacterial cells. This study demonstrated the effective utilisation of a long carbon chain alkanediol, namely 1,8-octanediol, to enhance the yield and production of a copolymer with a high molecular weight of over 1000 kDa, which is desirable for novel applications in medical and biopharmaceuticals. The increased PHA content (47-61 wt%) and concentration (1.7-4.5 g/L) was achieved by additional feeding of a combination of C4 substrates at C/N 10, with 1,8-octanediol + γ-butyrolactone producing P(3HB-co-22 mol% 4HB) with a high molecular weight (1060 kDa) and elongation at break of 970%. The DO-stat feeding strategy of C/N 10 has shown an increment of PHA concentration for both carbon combination, 0.45-4.27 g/L and 0.32-3.36 g/L for 1,8-octanediol + sodium 4-hydroxybutyrate (4HB-Na) and 1,8-octanediol + γ-butyrolactone, but with a slight reduction on molecular weight and mechanical strength. Nonetheless, further study revealed that a nitrogen-absence feeding strategy could retain the high molecular weight and elongation at break of the copolymer, and simultaneously improving the overall P(3HB-co-4HB) production.
Polyhydroxyalkanoate (PHA) is a biogenic polymer that has the potential to substitute synthetic plastic in numerous applications. This is due to its unique attribute of being a biodegradable and biocompatible thermoplastic, achievable through microbial fermentation from a broad utilizable range of renewable resources. Among all the PHAs discovered, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] stands out as a next generation healthcare biomaterial for having high biopharmaceutical and medical value since it is highly compatible to mammalian tissue. This review provides a critical assessment and complete overview of the development and trend of P(3HB-co-4HB) research over the last few decades, highlighting aspects from the microbial strain discovery to metabolic engineering and bioprocess cultivation strategies. The article also outlines the relevance of P(3HB-co-4HB) as a material for high value-added products in numerous healthcare-related applications.
Bacterial classification on the basis of a polyphasic approach was conducted on three poly(3 hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] accumulating bacterial strains that were isolated from samples collected from Malaysian environments; Kulim Lake, Sg. Pinang river and Sg. Manik paddy field. The Gram-negative, rod-shaped, motile, non-sporulating and non-fermenting bacteria were shown to belong to the genus Cupriavidus of the Betaproteobacteria on the basis of their 16S rRNA gene sequence analyses. The sequence similarity value with their near phylogenetic neighbour, Cupriavidus pauculus LMG3413T, was 98.5%. However, the DNA-DNA hybridization values (8-58%) and ribotyping analysis both enabled these strains to be differentiated from related Cupriavidus species with validly published names. The RiboPrint patterns of the three strains also revealed that the strains were genetically related even though they displayed a clonal diversity. The major cellular fatty acids detected in these strains included C15:0 ISO 2OH/C16:1 ω7c, hexadecanoic (16:0) and cis-11-octadecenoic (C18:1 ω7c). Their G+C contents ranged from 68.0 to 68.6 mol%, and their major isoprenoid quinone was Ubiquinone Q-8. Of these three strains, only strain USMAHM13 (= DSM 25816 = KCTC 32390) was discovered to exhibit yellow pigmentation that is characteristic of the carotenoid family. Their assembled genomes also showed that the three strains were not identical in terms of their genome sizes that were 7.82, 7.95 and 8.70 Mb for strains USMAHM13, USMAA1020 and USMAA2-4, respectively, which are slightly larger than that of Cupriavidus necator H16 (7.42 Mb). The average nucleotide identity (ANI) results indicated that the strains were genetically related and the genome pairs belong to the same species. On the basis of the results obtained in this study, the three strains are considered to represent a novel species for which the name Cupriavidus malaysiensis sp. nov. is proposed. The type strain of the species is USMAA1020T (= DSM 19416T = KCTC 32390T).
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
A locally isolated soil microorganism identified as Erwinia sp. USMI-20 has been found to produce poly(3-hydroxybutyrate), P(3HB), from either palm oil or glucose and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), from a combination of palm oil and a second carbon source of either one of the following compounds: propionic acid, n-propanol, valeric acid and n-pentanol. It was found that Erwinia sp. USMI-20 could produce P(3HB) up to 69 wt.% polymer content with a dry cell weight of 4.4 g/l from an initial amount of 14.5 g/l of glucose followed by a feeding rate of glucose at 0.48 g/h glucose. On the other hand, the bacteria can achieve 46 wt.% of P(3HB) and a dry cell weight of 3.6 g/l from a batch fermentation in a 10-l fermentor from an initial concentration of 4.6 g/l of palm oil. Further characterisation of the polymer production was also carried out by using different types of palm oil. Among the different palm oils that were used, crude palm oil was the best lipid source for P(3HB) production as compared to palm olein and palm kernel oil. In the production of the copolymer, P(3HB-co-3HV), the highest mole fraction of 3-HV units could be as high as 47 mol% from a single feeding of valeric acid upon initial growth on palm oil.
Multi-component nanohybrids comprising of organo-modified montmorillonite (MMT) and immiscible biopolymer blends of poly(3-hydroxybutyrate) (PHB) and epoxidized natural rubber (ENR-50) were prepared by solvent casting technique. The one and three dimensional morphology of PHB/ENR-50/MMT systems were studied using Polarizing Optical Microscopy (POM) and Scanning Electron Microscopy (SEM). Differential scanning calorimetry (DSC) technique was used to evaluate the thermal properties of the nanohybrids. The melting temperature (Tm) and enthalpy of melting (ΔHm) of PHB decrease with respect to the increase in ENR-50 as well as MMT content. The non-isothermal decomposition of the nanohybrids was studied using thermogravimetric (TG-DTG) analysis. FTIR-ATR spectra supported ring opening of the epoxide group via reaction with carboxyl group of PHB and amines of organic modifier. The reaction mechanism towards the formation of the nanohybrids is proposed.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is noted for its high biocompatibility, which makes it an excellent candidate for biopharmaceutical applications. The wild-type Cupriavidus sp. USMAA1020 strain is able to synthesize P(3HB-co-4HB) copolymers with different 4HB monomer compositions (up to 70mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and γ-butyrolactone were applied for the production of P(3HB-co-4HB) with different 4HB molar fraction. A sharp increase in 4HB monomer composition was attained by introducing additional copies of PHA synthase gene (phaC), responsible for P(3HB-co-4HB) polymerization. The phaC of Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 were cloned and heterologously introduced into host, wild-type Cupriavidus sp. USMAA1020. The gene dosage treatment resulted in the accumulation of 93mol% 4HB by the transformant strains when grown in similar conditions as the wild-type USMAA1020. The PHA synthase activities for both transformants were almost two-fold higher than the wild-type. The ability of the transformants to produce copolymers with high 4HB monomer composition was also tested in large scale production system using 5L and 30L bioreactors with a constant oxygen mass transfer rate. The 4HB monomer composition could be maintained at a range of 83-89mol%. The mechanical and thermal properties of copolymers improved with increasing 4HB monomer composition. The copolymers produced could be tailored for specific biopharmaceutical applications based on their properties.
Surface modification of superparamagnetic iron oxide nanoparticles (SPIONs) has been introduced with lauric acid and oleic acid via co-precipitation and thermal decomposition methods, respectively. This modification is required to increase the stability of SPIONs when incorporated in hydrophobic, biodegradable and biocompatible polymers such as poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In this work, the solid-in-oil-in-water (S/O/W) emulsion-solvent extraction/evaporation method was utilized to fabricate magnetic polymer microspheres incorporating SPIONs in PHBV. The prepared magnetic PHBV microspheres exhibited particle sizes <1 µm. The presence of functional groups of lauric acid, oleic acid and iron oxide in the PHBV microspheres was confirmed by Fourier Transform Infrared spectroscopy (FTIR). X-ray diffraction (XRD) analysis was performed to further confirm the success of the combination of modified SPIONs and PHBV. Thermogravimetric analysis (TGA) indicated that PHBV microspheres were incorporated with SPIONsLauric as compared with SPIONsOleic. This was also proven via magnetic susceptibility measurement as a higher value of this magnetic property was detected for PHBV/SPIONsLauric microspheres. It was revealed that the magnetic PHBV microspheres were non-toxic when assessed with mouse embryotic fibroblast cells (MEF) at different concentrations of microspheres. These results confirmed that the fabricated magnetic PHBV microspheres are potential candidates for use in biomedical applications.
The osteogenic potential of human adipose-derived stem cells (HADSCs) co-cultured with human osteoblasts (HOBs) using selected HADSCs/HOBs ratios of 1:1, 2:1, and 1:2, respectively, is evaluated. The HADSCs/HOBs were seeded on electrospun three-dimensional poly[(R)-3-hydroxybutyric acid] (PHB) blended with bovine-derived hydroxyapatite (BHA). Monocultures of HADSCs and HOBs were used as control groups. The effects of PHB-BHA scaffold on cell proliferation and cell morphology were assessed by AlamarBlue assay and field emission scanning electron microscopy. Cell differentiation, cell mineralization, and osteogenic-related gene expression of co-culture HADSCs/HOBs were examined by alkaline phosphatase (ALP) assay, alizarin Red S assay, and quantitative real time PCR, respectively. The results showed that co-culture of HADSCs/HOBs, 1:1 grown into PHB-BHA promoted better cell adhesion, displayed a significant higher cell proliferation, higher production of ALP, extracellular mineralization and osteogenic-related gene expression of run-related transcription factor, bone sialoprotein, osteopontin, and osteocalcin compared to other co-culture groups. This result also suggests that the use of electrospun PHB-BHA in a co-culture HADSCs/HOBs system may serve as promising approach to facilitate osteogenic differentiation activity of HADSCs through direct cell-to-cell contact with HOBs.