In vitro biocompatibility and degradation of terpolyester 3HB-co-4HB-co-3HHx,consisting of 3-hydroxybutyrate, 4-hydroxybutyrate and 3-hydroxyhexanoate

A terpolyester consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyhexanoate (3HHx), abbreviated as P(3HB-co-4HB-co-3HHx), was studied for possible application as an implant biomaterial. L929 mouse fibroblasts, MC3T3-E1 murine osteoblasts and a human cell line of immortalized human keratinocyte (HaCat cells) were used to study the biocompatibility of P(3HB-co-4HB-co-3HHx). Cell morphology and cell activity were studied using scanning electron microscopy (SEM) and the MTT assay, respectively. All three cell types showed higher activities when grown on films of P(3HB-co-4HB-co-3HHx) compared with their growth on poly(lactic acid) (PLA), co-polyester PHBHHx films and on polylysine-coated plates (blank), respectively. The three cell types grown on the terpolyester also demonstrated a well-spread cell shape and large number of pseudopods due to strong cell–cell and cell–material interactions. It was clearly observed that P(3HB-co-4HB-co-3HHx) had a much faster degradation rate than PHBHHx after 15 weeks of incubation in phosphate-buffered saline under dynamic conditions. The results proved that the terpolyester had favorable biocompatibility and biodegradability compared with the well-studied polyesters PLA and PHBHHx.     

Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only β-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.     

Physical Properties and Biocompatibility of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Blended with Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was blended with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) to improve physical properties and biocompatibility of PHBHHx for a wide range of biomedical applications. PHBHHx was completely miscible with P3HB4HB in their blends. All the PHBHHx/P3HB4HB blends showed improved physical properties compared with PHBHHx, including higher thermal stability, flexibility and mechanical strength. All the blends had more hydrophilic surface, higher polar component and rougher surface than PHBHHx. The PHBHHx/P3HB4HB blend in 4:2 weight ratio showed the roughest surface and also had the highest chondrocyte viability among all the blends and the polymers tested, which was 59% higher than that on PHBHHx and 32% higher than that on P3HB4HB. The blend with 4:2 weight ratio also had the maximum cartilage-specific collagen II mRNA expression among all the blends and the polymers tested, which was 9-times higher than that on PHBHHx and 8-times higher than that on P3HB4HB. These results demonstrated that PHBHHx had improved physical properties and biocompatibility after blending with P3HB4HB. The blends could be used for cartilage tissue engineering.     

Synthesis,Characterization and Cell Compatibility of Novel Poly(ester urethane)s Based on Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Prepared by Melting Polymerization

Novel tailor-made poly(ester urethane)s (PUs) based on microbial polyesters poly(3-hydroxybutyrate-co-4hydroxybutyrate) (P3HB4HB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) were synthesized by melting polymerization (MP) using 1,6-hexamethylene diisocyanate (HDI) as a coupling agent. A comprehensive characterization using ¹H-NMR, Fourier transform infrared spectroscopy (FT-IR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), mechanical properties, static water contact angles, cell proliferation using smooth muscle cells from rabbit aorta (RaSMCs) and immortalized human keratinocytes (HaCat), and blood coagulation behavior were conducted on the synthesized PUs films. DSC showed that PU samples had a low degree of crystallinity at room temperature and became fully amorphous after a melt-quenched process. The series of tailor-made PUs based on different mass ratios of P3HB4HB and PHBHHx revealed a ductile and flexile mechanical property especially for PHBHHx-rich PU, or a hydrophobic property for 4HB-rich PU. A 4 days incubation experiment showed that all PU films had a better cell proliferation than poly(lactic acid) (PLA), polyhydroxybutyrate (PHB), P3HB4HB and PHBHHx. RaSMCs cultured on PU films had a quiescent contractile phenotype, indicating that they were fully functional. HaCat incubated on tailor-made PU films showed a proliferation approximately equal to tissue-culture plates (TCPs). Blood coagulation behavior tests revealed a strong platelet adhesion and a short coagulation time on PU films. This study demonstrated potential medical applications for P3HB4HB and PHBHHx based polyurethane as a hydrophobic wound-healing and hemostatic materials.     

Effect of 3-hydroxyhexanoate content in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) on in vitro growth and differentiation of smooth muscle cells

In this paper, comprehensive characteristics including cell attachment, cell proliferation status, cell cycle progression and phenotypic changes of smooth muscle cells from rabbit aorta (RaSMCs) were studied on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 0-20% HHx (mol%) in comparison with tissue culture plates (TCPs). Results demonstrated that RaSMCs adhered better on PHBHHx containing 12% HHx (12%HHx) although they proliferated better on 20%HHx-containing PHBHHx films (20%HHx). This was explained by the difference in cell cycle progression observed using flow cytometry, as it was found that only 20%HHx-containing polymer could maintain the normal cell cycle evolution as TCPs did after 3 d incubation. The highest expression level and typical spindle-like distribution of alpha-actin on 20%HHx-containing polymer were characterized as the contractile-like phenotype, suggesting that RaSMCs tended to differentiate rather than proliferate compared to the cells grown on 12%HHx polymer. Results obtained above suggested that 20%HHx was suitable for RaSMCs proliferation, leading to its change to contractile phenotype. This study extends the potential applications of PHBHHx in SMCs-related graft scaffold fabrication for tissue engineering.     

Hydroxyapatite reinforced poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) based degradable composite bone plate

Poly(3-hydroxybutyrate) (P3HB), its co-polymers with 3-hydroxyvalerate (HV) (PHBV8 and PHBV22), and their hydroxyapatite (HAp) containing composites (5 and 15%, w/w) were prepared by injection molding. PHBV bone plates with low valerate contents and 15% (w/w) HAp appear to have better mechanical properties than the others. Flexural strengths of 15% (w/w) HAp-loaded P3HB, PHBV8 and PHBV22 were 78.28, 63.45 and 39.38 MPa, respectively. Tensile strengths of 15% (w/w) HAp-loaded P3HB, PHBV8 and PHBV22 were 18.99, 15.44 and 11.02 MPa, respectively. For the ageing test, bone plates were incubated in phosphate-buffered saline PBS (0.1 M, pH 7.4) at 37°C and at pre-determined time points they were removed and subjected to a three-point bending test. Incubation in PBS caused a sharp decrease in the mechanical properties within the first 24 h, followed either by a gradual decrease or no change for a period of about 1 month. SEM results showed that there was no significant material erosion in the 4-week incubation period. P3HB loaded with 15% HAp appeared to yield the most suitable bone plate, insofar as mechanical properties are concerned with potential for further testing in vivo.     

Properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films modified with polyvinylpyrrolidone and behavior of MC3T3-E1 osteoblasts cultured on the blended films

A series of composite films of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) modified with polyvinylpyrrolidone (PVP) was prepared by varying the ratio of constituents, and their properties and cytocompatibility were evaluated. The hydrophilicity of the blended materials surfaces increased and the amounts of fibronectin and laminin adsorbed on the materials surface increased remarkably compared with PHBHHx. FT-IR spectra of the blended films showed a new band, implying that a surface physical interpenetrating network structure had formed. Scanning electron microscopy showed that there were dense pits and holes on the blended films surface. For the films of PHBHHx with 20 wt% and 40 wt% PVP, MTT assay indicated that PVP enhanced cell adhesion and proliferation, but that the effects were impaired by excessive PVP. The results suggested that proper addition of PVP increased the cytocompatibility of PHBHHx because the material surface had increased hydrophilicity and presented an appropriate morphology.     

Accelerating and increasing nano-scaled pore formation on electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers

Porous fibers are advantageous for filtration systems, drug delivery systems, and in the field of tissue engineering, in comparison to their non-porous counterparts. In this study, we developed a facile technique including two steps to generate poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) porous fibers with a controllable pore size. An electrospinning technique was employed to obtain five types of PHBV/poly(ethylene oxide) (PEO)-blended fibers (PHBV:PEO = 9:1, 8:2, 7:3, 6:4, 5:5) with PEO as the porogen. PEO was leached out by simulated body fluid (SBF) and water, respectively. The pore morphology and calcium deposition of the resulting fibers were compared to those formed on film through the SEM-EDX analysis. It was revealed that pore size and number increased with increasing PEO percentage in the fiber or film. The pore size on the films (at micrometer scale) was much larger than that of nanofibers, which was in the range of 70–120 nm. The simultaneous removal of PEO and deposition of calcium phosphate through SBF buffer enhanced synergistically both the pore formation and mineral deposition. The different phase separation mechanisms explain the different pore morphologies in the film and the nanofibers. The cellular experimental results show that fibers with nanometer-scale pores and minerals can enhance the proliferation of bone marrow-derived mesenchymal stem cells.     

The mechanical properties and in vitro biodegradation and biocompatibility of UV-treated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Strong mechanical properties and controllable biodegradability, together with biocompatibility, are the important requirement for the development of medical implant materials. In this study, an ultraviolet (UV) radiation method was developed to achieve controlled degradation for bacterial biopolyester poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) which has a low biodegradation rate that limits its application for many implant applications required quick degradation. When UV radiation was applied directly to PHBHHx powder, significant molecular weight (Mw) losses were observed with the powder, Mw reduction depended on the UV radiation time. At the same time, a broad PHBHHx Mw distribution was the result of inhomogeneous radiation. Interestingly, this inhomogeneous radiation helped maintain the mechanical properties of films made of the UV-radiated powder. In comparison, the PHBHHx films subjected to direct UV radiation became very brittle although their degradation was faster than that of the PHBHHx powders subjected to direct UV radiation. After 15 weeks of degradation in simulated body fluid (SBF), films prepared from 8 and 16h UV-treated PHBHHx powders maintained 92% and 87% of their original weights, respectively, while the untreated PHBHHx films lost only 1% of its weight. Significant increases in growth of fibroblast L929 were observed on films prepared from UV-radiated powders. This improved biocompatibility could be attributed to increasing hydrophilic functional groups generated by increasing polar groups C-O and CO. In general, UV-treated PHBHHx powder had a broad Mw distribution, which contributed to fast degradation due to dissolution of low Mw polymer fragments, and strong mechanical property due to high Mw polymer chains. Combined with its improved biocompatibility, PHBHHx is one more step close to become a biomedical implant material.     

In vivo studies of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) based polymers: biodegradation and tissue reactions

The in vivo tissue reactions and biodegradations of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(lactide) (PLA), poly(3-hydroxybutyrate) (PHB), blends of PHBHHx (X) and poly(ethylene glycol) (PEG) (E) with ratios of 1:1 (E1X1) and 1:5 (E1X5), respectively, were evaluated by subcutaneous implantation in rabbits. Results revealed that the degradation rate increased in the order of PHB < PHBHHx < PLA. During the implantation period, crystallinity of PHBHHx increased from 19% to 22% and then dropped to 14%. Gel permeation chromatography (GPC) displayed increasing polydispersity and typical bimodal distribution from 3 to 6 months. The above results suggested that rapid PHBHHx degradation occurred in amorphous region rather than in crystalline region. While the in vivo hydrolysis of PHB was found to start from a random chain scission both in amorphous and crystalline regions of the polymer matrix, as demonstrated by its hydrolysis process accompanied by a decrease in molecular weight with unimodal distribution and relatively narrow polydispersity. Compared to pure PHBHHx, PHBHHx-PEG blends showed accelerated weight loss of PHBHHx with weak molecular weight reduction. In general, PHBHHx elicited a very mild tissue response during implantation lasting 6 months compared with relative acute immunological reactions observed among PHB and PLA objects, respectively. Pronounced tissue responses were observed in the capsule surrounding E1X1 and E1X5 as characterized by the presence of lymphocytes, eosinophils and vascularization, which might be resulted from the continuous leaching of PEG.     

Studies on bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)

The objective of this study is to investigate the biocompatibility of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with bone marrow stromal cells in vitro. The adsorption of fibronectin on the material was studied by enzyme-linked immunosorbent assay. After bone marrow stromal cells were seeded and cultured on PHBHHx, their proliferation was investigated by MTT. Differentiation of the cells was assessed by measuring alkaline phosphatase activity and by histochemical assay. The wettability and thermal property of PHBHHx films were also studied by contact angle goniometer, thermogravimetry and differential scanning calorimetry, respectively. The results show that bone marrow stromal cells can attach, proliferate and differentiate into osteoblasts on PHBHHx films. These results suggest that PHBHHx has good affinity with bone marrow stromal cells and may have potential applications in bone tissue engineering.     

Study on the biocompatibility of novel terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate)

Terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) containing 5.3 mol % 3-hydroxyvalerate (3-HV) and 10.2 mol % 3-hydroxyhexanoate was obtained via microbial synthesis using recombinant Aeromonas hydrophila. For the first time in vitro biocompatibility of the terpolyester was evaluated in comparison with poly(L-lactic acid), poly (3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Scanning electron microscopy showed whorls-like morphology only on PHBVHHx film surface. Methylthiazol tetrazolium assay demonstrated that PHBVHHx was better than the above four materials in promoting cell attachment and proliferation of fibroblast cell line L929 and osteoblast cell line MC3T3, respectively. Histological study using rabbits proved that PHBVHHx was a fairly harmless implantable biomaterial. Thus, PHBVHHx with an adjustable mechanical properties, combined with its biocompatibility, are in the process of developing into a new generation of bioimplant material.     

Preparation and evaluation of porous poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) hydroxyapatite composite scaffolds

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and PHBHHx-hydroxyapatite (HAP) composite scaffolds have been prepared by phase separation and subsequent sublimation of the solvent for bone tissue engineering. Scanning electron microscopy (SEM), porosity measurement, mechanical tests, and thermogravimertric analysis (TGA) are used to analyze the physical properties of the scaffolds. The biocompatibility and osteoconductivity are assessed by examining the morphology, proliferation, and differentiation of MC3T3-E1 osteoprogenitor cells seeded on the scaffolds. The PHBHHx-HAP composite scaffolds show better mechanical properties, biocompatibility, and osteoconductivity than the PHBHHx scaffolds. The results suggest that PHBHHx-HAP composite scaffolds can be employed as a promising candidate for bone reconstruction.     

Evaluation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) conduits for peripheral nerve regeneration

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was investigated for possible application in repairing damaged nerves. Porous nerve conduits with both uniform wall porosity and non-uniform wall porosity were prepared using a particle leaching method. Adult Sprague-Dawley (SD) rats weighing 200-250 g were used as the animal model. The conduits were employed to bridge the 10mm defects in the sciatic nerve of the Sprague-Dawley (SD) rats. Mechanical tests showed that the PHBHHx nerve conduits had proper mechanical properties including maximal loads of 3.1N and 1.3N for the conduits with non-uniform wall porosity and with uniform wall porosity, respectively, and maximal stresses of 2.3 MPa and 0.94 MPa for the conduits with non-uniform wall porosity and with uniform wall porosity, respectively. At the same time, both types of conduits were permeable to three compounds tested including glucose, lysozyme and bovine serum albumin, indicating the suitability of the conduits for free exchanges of nutrients. Compound Muscle Action Potentials (CMAPs) were clearly observed in both types of the PHBHHx nerve conduits after 1 month of implantation, indicating a rapid functional recovery for the disrupted nerves. The results of histological sections demonstrated that the internal sides of the conduits with non-uniform wall porosity were compact enough to prevent the connective tissues from ingrowth penetration. After implantation for 3 months in the rats, the conduits with uniform wall porosity and those with non-uniform wall porosity lost 24% and 20% of their original weight average molecular weights, respectively. Combined with the strong mechanical properties, good nerve regeneration ability and non-toxicity of its degradation products, PHBHHx nerve conduits can be developed into a useful material to repair nerve damage.     

Effect of composition of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) on growth of fibroblast and osteoblast

Films made of poly (3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx) consisting of 5%, 12% and 20% hydroxyhexanoate (HHx), respectively, were evaluated for biomedical application in comparison with poly (L-Lactide) (PLA). With the increase of HHx content in PHBHHx, the polymer surface properties changed accordingly. P(HB-co-20%-HHx) had the smoothest surface while PHB surface was most hydrophilic among the evaluated PHB and all the PHBHHx. All PHBHHx also showed strong protein affinity and biocompatibility. It was found that fibroblast and osteoblast had different responses to these polymers: fibroblast cells favored P(HB-co-20%-HHx), yet osteoblast cells preferred P(HB-co-12%-HHx). PHB and all PHBHHx appeared to have better biocompatibility for fibroblast and osteoblast compared with PLA. Polymers possessing different surface properties may help meet different cellular requirements. Combined with their good mechanical properties for elongation and adjustable biocompatibility, PHBHHx may meet the needs of growth requirements of different tissues and cells.     

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.     

Interactions between a poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) terpolyester and human keratinocytes

A new member of polyhydroxyalkanoates (PHA) family, namely, a terpolyester abbreviated as PHBVHHx consisting of 3-hydroxybutyrate (HB), 3-hydroxyvalerate (HV) and 3-hydroxyhexanoate (HHx) that can be produced by recombinant microorganisms, was found to have proper thermo- and mechanical properties for possible skin tissue engineering, as demonstrated by its strong ability to support the growth of human keratinocyte cell line HaCaT. In this study, HaCaT cells showed the strongest viability and the highest growth activity on PHBVHHx film compared with PLA, PHB, PHBV, PHBHHx and P3HB4HB, even the tissue culture plates were grown with less HaCaT cells compared with that on PHBVHHx. To understand its superior biocompatibility, PHBVHHx nanoparticles ranging from 200 to 350nm were prepared. It was found that the nanoparticles could increase the cellular activities by stimulating a rapid increase of cytosolic calcium influx in HaCaT cells, leading to enhanced cell growth. At the same time, 3-hydroxybutyrate (HB), a degradation product and the main component of PHBVHHx, was also shown to promote HaCaT proliferation. Morphologically, under the same preparation conditions, PHBVHHx film showed the most obvious surface roughness under atomic force microscopy (AFM), accompanied by the lowest surface energy compared with all other well studied biopolymers tested above. These results explained the superior ability for PHBVHHx to grow skin HaCaT cells. Therefore, PHBVHHx possesses the suitability to be developed into a skin tissue-engineered material.     

Attachment, proliferation and differentiation of osteoblasts on random biopolyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds

Rabbit bone marrow cells were inoculated on 3D scaffolds of poly(lactic acid) (PLA), poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) to evaluate their in vitro biocompatibilities. It was found that PHBHHx had the best performance on attachment, proliferation of bone marrow cells. The cells on PHBHHx scaffolds presented typical osteoblast phenotypes: round cell shape, high alkaline phosphotase (ALP) activity, strong calcium deposition, and fibrillar collagen synthesis. After incubation for 10 days, cells grown on PHBHHx scaffolds were approximately 2x10(5)ml(-1), 40% more than that on PHB scaffolds and 60% more than that on PLA scaffolds. ALP activity of the cells grown on PHBHHx scaffolds was up to about 65U/g scaffolds, 50% higher than that of PHB and PLA, respectively. The scanning electronic microscopy (SEM) results showed that PHBHHx scaffolds had the appropriate roughness for osteoblast attachment and proliferation comparing with PHB and PLA. All these indicated that PHBHHx was a suitable biomaterial for osteoblast attachment, proliferation and differentiation from bone marrow cells.     

In vitro investigation of maleated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for its biocompatibility to mouse fibroblast L929 and human microvascular endothelial cells

Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a polyester with strong mechanical properties and biocompatibility, is a member of microbial polyhydroxyalkanoates (PHA) family. Maleic anhydride was used to graft PHBHHx to form maleated PHBHHx (Ma-PHBHHx). Ma-PHBHHx with a graft degree of 0.59% was found to be more thermo-stable in comparison with PHBHHx. In vitro study demonstrated that the biocompatibility to mouse fibroblast L929 and human microvascular endothelial cells (HMEC) was improved in different degrees on Ma-PHBHHx films and scaffolds. Compared with their growth on PHBHHx, L929 and HMEC grown on Ma-PHBHHx films and scaffolds showed approximately 120% and 260% more in proliferation rates, respectively. Morphology study suggested that fine whorl-like surface structures with porosities on Ma-PHBHHx films attributed to MA grafting would be better for cell attachment and proliferation. Ma-PHBHHx scaffolds prepared by thermally induced phase separation (TIPS) with increased porosity, hydrophilicity, surface energy, and charges also were more favorable for cell growth. In addition, Ma-PHBHHx showed an accelerated degradation incubation in SBF at 37 degrees C, losing 21.4% of its original weight after 21 weeks while PHBHHx just lost 7.3%. Based on the improved biocompatibility, reasonable mechanical properties as well as accelerated biodegradation, Ma-PHBHHx has shown advantages over PHBHHx as a biomaterial for biomedical applications.     

Influence of poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) on growth and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

As a new member of polyhydroxyalkanoate (PHA) family, the novel polyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-4HB-3HHx)) was produced by recombinant Aeromonas hydrophila 4AK4 and used for the first time to test its biocompatibility. It was shown that P(3HB-4HB-3HHx) had higher hydrophobicity, surface energy, and rougher surface than the well-studied polymers poly(L-lactic acid) (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Human mesenchymal stem cells (MSCs) attached better on P(3HB-4HB-3HHx) film than on tissue culture plates (TCPs), PLA film, and PHBHHx film. MSC proliferation on P(3HB-4HB-3HHx) film was 126% higher than that on TCPs, 84% higher than that on PHBHHx film, and 312% higher than that on PLA film (p < 0.01). P(3HB-4HB-3HHx) also supported osteogenic differentiation of MSCs. Previous studies found that all PHA materials tested were either less than or equal to TCPs for supporting cell growth. Among all PHA materials tested, P(3HB-4HB-3HHx) was the only PHA material to significantly promote cell proliferation compared with TCPs. P(3HB-4HB-3HHx) could be exploited for applications in bone tissue engineering.