Guo-Qiang Chen

Polyhydroxyalkanoate (PHA) scaffolds with good mechanical properties and biocompatibility

Blending microbial polyesters polyhydroxyalkanoates containing polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) were turned into films and scaffolds. The films made from blending polyesters showed that the elongation to break of the blending PHBHHx/PHB film increased from 15% to 106% when PHBHHx content in the blend increased from 40% to 60%. Scaffolds made of PHBHHx/PHB consisting of 60 wt% PHBHHx showed strong growth and proliferation of chondrocytes on the blending materials under scanning electron microscope. Energy dispersive X-ray analysis of the extra cellular matrix on the scaffolds demonstrated a high level of calcium and phosphorus elements in a molar ratio of Ca/P at 1.66, this is approximately equal to that of natural material hydroxyapatite which has a Ca/P ratio of 1.67. This suggested that the chondrocyte cells grown on PHBHHx/PHB scaffolds presented effective physiological function for generation of cartilage.

Effect of surface treatment on the biocompatibility of microbial polyhydroxyalkanoates.

The biocompatibility of microbial polyesters polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) were evaluated in vitro. The mouse fibroblast cell line L929 was inoculated on films made of PHB, PHBHHx and their blends, polylactic acid (PLA) as control. It was found that the growth of the cells L929 was poor on PHB and PLA films. The viable cell number ranged from 8.8 x 10(2) to 1.8 x 10(4)/cm2 only. Cell growth on the films made by blending PHB and PHBHHx showed a dramatic improvement. The viable cell number observed increased from 9.7 x 10(2) to 1.9 x 10(5) on a series of PHB/PHBHHx blended film in ratios of 0.9/0.1:0/1, respectively, indicating a much better biocompatibility in the blends contributed by PHBHHx. Biocompatibility was also strongly improved when these polymers were treated with lipases and NaOH, respectively. However, the effects of treatment were weakened when PHBHHx content increased in the blends. It was found that lipase treatment had more increased biocompatibility than NaOH. After the treatment biocompatibility of PHB was approximately the same as PLA, while PHBHHx and its dominant blends showed improved biocompatibility compared to PLA.

Study on the three-dimensional proliferation of rabbit articular cartilage-derived chondrocytes on polyhydroxyalkanoate scaffolds

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx)/polyhydroxybutyrate (PHB) (PHBHHx/PHB) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture. Blend polymers of PHBHHx/PHB were fabricated into three-dimensional porous scaffolds by the salt-leaching method. Chondrocytes isolated from rabbit articular cartilage (RAC) were seeded on the scaffolds and incubated over 28 days, with change of the culture medium every 4 days. PHB scaffold was taken as a control. Methylthiazol tetrazolium (MTT) (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zolium bromide) assay was used to quantitatively examine the proliferation of chondrocytes. Results showed that chondrocytes proliferated better on the PHBHHx/PHB scaffolds than on PHB one. The maximal cell densities were all observed after 7 days of incubation. As for the blend polymers, cells grew better on scaffolds consisting of PHBHHx/PHB in ratios of 2:1 and 1:2 than they did on PHBHHx/PHB of 1:1. Scanning electron microscopy (SEM) also showed that large quantities of chondrocytes grew initially on the surface of the scaffold. After 7 days, they further grew into the open pores of the blend polymer scaffolds. Morphologically, cells found on the surface of the scaffold exhibited a flat appearance and slowly form confluent cell multilayers starting from 14 to 28 days of the growth. In contrast, cells showed rounded morphology, formed aggregates and islets inside the scaffolds. In addition, chondrocytes proliferated on the scaffold and preserved their phenotype for up to 28 days.

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.

A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy

Abstract Polyhydroxyalkanoates (PHA) are synthesized by many bacteria as inclusion bodies, and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. In this paper, Fourier-transform infrared spectroscopy (FT-IR) was used to carry out rapid qualitative analysis of PHA in intact bacterial cells. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mclHA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, showed their strong characteristic band at 1728 cm−1, 1740 cm−1 or 1732 cm−1 respectively. Other accompanying bands near 1280 cm−1 and 1165 cm−1 helped identify the types of PHA. The intensity of the methylene band near 2925 cm−1 provided additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm−1, 1744 cm−1 or 1739 cm−1, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. The FT-IR results were further confirmed by gas chromatography analysis.

Polyhydroxyalkanoates as a source of chemicals, polymers, and biofuels.

Microbial polyhydroxyalkanoates (PHA) are a family of structurally diverse polyesters produced by many bacteria. Deleting key steps from the beta-oxidation cycle in Pseudomonas putida makes it possible to achieve precise substrate based design of PHA homopolymers, copolymers, and block polymers, allowing the study of structure-property relationship in a clear way. The PHA homopolymer synthesis also allows the microbial or chemical production of pure monomers of PHA in a convenient way without separating the mixed monomers. After used as bioplastics, PHA can be methyl esterified to become biofuels, which further extends the PHA application value. The microbial production of PHA with diverse structures is entering a new developing phase.

Gelatin nanofibrous membrane fabricated by electrospinning of aqueous gelatin solution for guided tissue regeneration

The electrospinning of gelatin aqueous solution was successfully carried out by elevating the spinning temperature. The effects of spinning temperature and solution concentration were investigated on the morphology of gelatin nanofibers in the current study. To improve the stability and mechanical properties in moist state, the gelatin nanofibrous membrane was chemically crosslinked by 1-ethyl-3-dimethyl-aminopropyl carbodiimide hydrochloride and N-hydroxyl succinimide. The concentration of crosslinker was optimized by measuring the swelling degree and weight loss. Nanofibrous structure of the membrane was retained after lyophilization, although the fibers were curled and conglutinated. Tensile test revealed that the hydrated membrane becomes pliable and provides predetermined mechanical properties. Periodontal ligament cells cultured on the membrane in vitro exhibited good cell attachment, growth, and proliferation. Gelatin nanofibrous membrane can be one of promising biomaterials for the regeneration of damaged periodontal tissues.

Reduced mouse fibroblast cell growth by increased hydrophilicity of microbial polyhydroxyalkanoates via hyaluronan coating.

The mouse fibroblast cell line L929 was inoculated on 3D scaffolds of microbial polyesters, namely polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) to evaluate their in vitro biocompatibility. It was found that both polyhydroxyalkanoates (PHA) subjected to lipase treatment and hyaluronan (HA) coating decreased the contact angle of water to the material surface approximately 30%, meaning an increased hydrophilicity on the PHA surface. At the same time, both the lipase treatment and the HA coating smoothened the PHA surface. After the lipase treatment or HA coating, the ratio of PHA hydrophilic groups including hydroxyl and carboxyl to carbonyl of PHA was approximately 1:1 or 2:1. Cells grown on scaffolds treated with lipase were approximately 4 x 10(5)/ml, twice in number of the control. However, PHA scaffolds coated with HA were observed with a 40% decrease in cell growth compared with that of the control. HA coating reduced the cell attachment and proliferation on PHA although the materials had increased hydrophilicity. In comparison, lipase treatment promoted the cell growth on PHA although the treatment did not lead to better hydrophilicity compared with HA coating. It appeared that an appropriate combination of hydrophilicity and hydrophobicity was important for the biocompatibility of PHBHHx, especially for the growth of L929 cells on the surface of this material. This may have instructive significance for biomaterial selection and design.

Medical Application of Microbial Biopolyesters Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) are a family of polyesters synthesized by microorganisms under unbalanced growth conditions. PHA including poly-3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly-4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and poly-3-hydroxyoctanoate (PHO) are now available in sufficient quantity for tissue engineering medical application studies due to their reasonable biocompatibility, adjustable mechanical properties, and controllable biodegradability. This paper reviews many achievements based on PHA for medical devices development, tissue repair, artificial organ construction, drug delivery, and nutritional/therapeutic uses. Combined with the recent FDA approval for P4HB clinical application, one can expect a good prospect for PHA application in the medical fields.

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.