Poly(hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of articular cartilage chondrocytes in vitro

The present investigation describes the production of extracellular matrix of rabbit articular cartilage chondrocytes grown on scaffolds of polyhydroxybutyrate (PHB) blended with poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for up to 7 days. The mRNA level of type II collagen of chondrocytes seeded on all scaffolds consisting of PHBHHx were obviously higher than that of PHB-only scaffold throughout the culture period, suggesting the positive effect of PHBHHx on extracellular matrix production. Second-harmonic generation (SHG) imaging technique, combined with confocal fluorescence microscopy (CFM) revealed that PHBHHx in PHB scaffold provided better surface properties for anchoring type II collagen filaments and their penetration into internal layers of the scaffolds. Glycosaminoglycan (GAG), a major composition of extracellular matrix, showed a sharp increase in construct of 1:2 PHB/PHBHHx scaffold after 7 day cultivation, while only a small increase was observed in all other tested scaffolds. At the same time, total collagen contents in all scaffolds containing PHBHHx increased with time, with the maximum collagen production of 742.1+/-99.2mg/g dry weight observed in construct of 1:2 PHB/PHBHHx scaffold inoculated for 7 days, this was almost 4-fold higher than that in scaffold of PHB only. It appears that the presence of right proportion of PHBHHx in the composite system of PHB/PHBHHx highly favored the production of extracellular matrix of articular cartilage chondrocytes.     

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.     

Effects of crystallization of polyhydroxyalkanoate blend on surface physicochemical properties and interactions with rabbit articular cartilage chondrocytes

As a new member of polyhydroxyalkanoate (PHA) family, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has better mechanical properties compared with poly(3-hydroxybutyrate) (PHB). Still, many properties of PHBHHx and its blend with PHB may be exploited for a wide range of applications, such as tissue engineering and drug delivery. In this study, PHBHHx was found to be completely miscible with PHB in their blends. Crystallization behavior of these polyester blends played an important role in determination of the surface physicochemical properties including surface free energy, chemical states and polarity, and could subsequently govern the cellular responses. Interaction between PHB and PHBHHx influenced the non-dispersion surface free energy component, which governed the total surface free energy. It was shown that the blend with a 1:1 ratio of PHB/PHBHHx possessed the highest surface free energy, which was the most optimal material for chondrocytes adhesion. After 24 h, the amount of chondrocytes adhered on films of PHB/PHBHHx (1:1) was 2.1 x 10(4)cells/cm(2), 5-times more compared with that on the PHB films (0.4 x 10(4)cells/cm(2)). The polarity of the blends increased with decreasing crystallinity. After 8 days of cultivation, the chondrocytes attached on PHB films were surrounded by both collagen II and collagen X, the amount of extracellular collagen X decreased with increasing polarity contributed by increasing PHBHHx content in the blend, while chondrocytes changed shapes from spherical to flat with increasing polarity. It indicated that endochondral ossification of chondrocytes was remarkably influenced by the crystallinity of the polyesters.     

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.     

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.     

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.     

PHB/PHBHHx scaffolds and human adipose-derived stem cells for cartilage tissue engineering

The goal of this study was to investigate the potential of polyhydroxybutyrate (PHB)/poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) (PHB/PHBHHx) to produce neocartilage upon seeding with differentiated human adipose-derived stem cells (hASCs). hASCs were grown on a three-dimensional PHB/PHBHHx scaffold in vitro with or without chondrogenic media for 14 days. Scanning electron microscopy showed that differentiated cells produced abundant extracellular matrices with increasing culture time. No cytotoxicity was observed by the live/dead cell viability assay. GAG and total collagen content in the differentiated cells increased significantly with in vitro culture time. After 14 days of in vitro culture, the differentiated cells grown on the (PHB/PHBHHx) scaffold (differentiated cells/(PHB/PHBHHx)) were implanted into the subcutaneous layer nude mice for 12 or 24 weeks, non-differentiated cells/(PHB/PHBHHx) were implanted as the control group. The differentiated cells/(PHB/PHBHHx) implants formed cartilage-like tissue after 24 weeks of implantation, and stained positive for collagen type II, safranin O, and toluidine blue. In addition, typical cartilage lacuna was observed, and there were no remnants of PHB/PHBHHx. Collagen type II was detected by Western blot at 12 and 24 weeks of implantation. In the control group, no cartilage formation was observed. This study demonstrated that PHB/PHBHHx is a suitable material for cartilage tissue engineering.     

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.     

Evaluation of three-dimensional scaffolds made of blends of hydroxyapatite and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for bone reconstruction

Hydroxyapatite (HAP) was blended into poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) to make films and scaffolds. After HAP blending, mechanical properties of PHB including compressive elastic modulus and maximum stress showed improvement and osteoblast responses including cell growth and alkaline phosphatase activity were also strengthened. On the other hand, scaffolds made of PHBHHx blended with HAP had an adverse effect. No remarkable change on degradation of PHB or PHBHHx blended with HAP, respectively, was observed in simulated body fluid. Scanning electron microscopy examination revealed that osteoblast responses to HAP incorporation may be related to surface morphology and to the exposed HAP particles on polymer surface. All these results indicated that the blending of HAP particles into PHBHHx scaffolds fabricated by salt leaching was unable to either strengthen its mechanical properties or enhance osteoblast responses. Although HAP is bioactive and osteoconductive, its blending with PHBHHx did not generate a better performance on bone reconstruction.     

Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide

Hydrophobic polyhydroxyalkanoate (PHA) scaffolds made of a copolyester of 3-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx) were coated with a fusion protein PHA granule binding protein PhaP fused with RGD peptide (PhaP-RGD). Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on/in the scaffolds for formation of articular cartilages derived from chondrogenic differentiation of hBMSCs for cartilage tissue engineering. PhaP-RGD coating led to more homogeneous spread of cells, better cell adhesion, proliferation and chondrogenic differentiation in the scaffolds compared with those of PhaP coated or uncoated scaffolds immerging in serum minus chondrogenic induction medium. In addition, more extracellular matrices were produced by the differentiated cells over a period of 14 days on/in the PhaP-RGD coated scaffolds evidenced by scanning electron microscopy imaging, enhanced expression of chondrocyte specific genes including SOX-9, aggrecan and type II collagen, suggesting the positive effect of RGD on extracellular matrix production. Furthermore, cartilage-specific extracellular substances sulphated glycosaminoglycans (sGAG) and total collagen content found on/in the PhaP-RGD coated scaffolds were significantly more compared with that produced by the control and PhaP only coated scaffolds. Homogeneously distributed chondrocytes-like cells forming cartilage-like matrices were observed on/in the PhaP-RGD coated scaffolds after 3 weeks. The results suggested that PhaP-RGD coated PHBHHx scaffold promoted chondrogenic differentiation of hBMSCs and could support cartilage tissue engineering.     

Application of Polyhydroxyalkanoates Nanoparticles as Intracellular Sustained Drug-Release Vectors

Polyhydroxybutyrate (PHB), co-polyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and polylactic acid (PLA) were used to prepare nanoparticles with average sizes of 160, 250 and 150 nm, respectively. A lipid-soluble colorant, rhodamine B isothiocyanate (RBITC), was employed to study drug-release behaviors from these nanoparticles. A high RBITC drug-loading efficiency of over 75% was achieved with all PHA nanoparticles prepared. Macrophage endocytosis led to an intracellular RBITC drug sustained release over a period of at least 20 days for PHB and PHBHHx nanoparticles, while PLA nanoparticles and free drug lasted only 15 days and a week, respectively. Polymer properties and particle sizes showed little effect on drug-release behavior. This study showed for the first time that PHB and PHBHHx can be used effectively to achieve intracellular controlled drug releases.     

Nanofibrous polyhydroxyalkanoate matrices as cell growth supporting materials

Polyhydroxyalkanoates (PHAs) have been demonstrated to be a family of biopolymers with good biodegradability and biocompatibility. To mimic the real microenvironment of extracellular matrix (ECM) for cell growth, novel nanofiber matrices based on PHA polymers were prepared via a phase separation process. Three-dimensional interconnected fibrous networks were observed in these matrices with average fiber diameters of 50-500 nm, which are very similar to the major ECM component collagen. Compared with nanofiber matrix made of poly(L-lactide), the mechanical properties of PHA nanofiber matrices were significantly improved, especially those matrices of PHA blends PHB/PHBHHx containing polyhydroxybutyrate (PHB) and copolyesters PHBHHx consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate, and PHB/P3HB4HB that are PHB blended with copolyesters of 3-hydroxybutyrate and 4-hydroxybutyrate, respectively. More importantly, cell attachment and growth of human keratinocyte cell line HaCat on the nanofiber PHA matrices showed a notable improvement over those on PHA matrices prepared via an ordinary solution casting method. It was therefore proposed that PHA nanofiber matrices combined the advantages of biodegradation, improved mechanical strengths and the nanostructure of a natural extracellular matrix, leading to a better cell compatibility, thus they can be used for future implant biomaterial development.     

Phase morphology, physical properties, and biodegradation behavior of novel PLA/PHBHHx blends

In this study, two biodegradable polyesters [i.e., polylactic acid (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)] with complementarity in terms of mechanical performance have been combined, and a series of blends with a broad range of compositions has been prepared by thermal compounding. The evolution of phase morphologies with the variation of compositions has been characterized by using Fourier transform infrared spectroscopic imaging together with scanning electron microscope analyses. Thermal, mechanical, and biodegradation properties of the PLA/PHBHHx blends were systematically investigated. Mechanical properties were further analyzed by using theoretical models and correlated with the results of the morphology/structure and compatibility of the blends. Results indicate that PLA/PHBHHx blends are immiscible but can be compatible to some extent at certain compositions (e.g., PLA/PHBHHx (w/w) = 80/20 and 20/80). The physical properties of the blend could be fine tuned by adjusting the blend composition.     

Fibrous Scaffolds Made by Co-Electrospinning Soluble Eggshell Membrane Protein with Biodegradable Synthetic Polymers

Abstract

Soluble eggshell membrane protein (SEP), isolated from natural eggshell membrane, was co-electrospun with biodegradable synthetic polymers poly(propylene carbonate) (PPC) and poly(lactic acid) (PLA) in various proportions from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solutions in order to prepare fibrous scaffolds having simultaneously good mechanical properties and biocompatibility. The fiber morphology was observed by field emission scanning electron microscopy, showing uniform fibers with diameter of 1.2–1.0 and 1.3–0.7 um for PPC/SEP and PLA/SEP blend fibers, respectively. Transmission electron microscopy observation shows that the blend fibers have domain-matrix phase morphology with fiber-like SEP domains in the PPC or PLA matrix, indicating the occurrence of phase separation, although interaction exists between PPC (or PLA) and SEP, as revealed by attenuated total reflectance Fourier transform infrared spectroscopy. The mechanical properties were evaluated by uniaxial tensile tests and showed that both the tensile strength and elongation at break increase with increasing incorporation of PPC (or PLA). The surface composition was investigated by X-ray photoelectron spectroscopy and SEP was found on the fiber surfaces, and as a result the surfaces of the fibrous scaffolds are superhydrophilic. NIH3T3 cell culture tests demonstrate that the PPC/SEP and PLA/SEP blend fibrous scaffolds have a much improved biocompatibility compared to pure PPC or PLA fibrous scaffolds.     

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.     

Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits

Articular cartilage repair using tissue engineering approach generally requires the use of an appropriate scaffold architecture that can support the formation of cartilage tissue. In this investigation, the potential of three-dimensional scaffolds made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated in rabbit articular cartilage defect model. Engineered PHBHHx cartilage constructs inoculated in vitro with rabbit chondrocytes for 30 days were examined. Subsequently the constructs inoculated with chondrocytes for 10 days were selected for transplantation into rabbits. After 16 weeks of in vivo implantation, both the engineered cartilage constructs and the bare scaffolds were found to be filled the defects with white cartilaginous tissue, with the engineered constructs showing histologically good subchondral bone connection and better surrounding cartilage infusion. Owing to pre-seeded chondrocytes in the PHBHHx scaffolds, better surface integrality and more accumulation of extracellular matrix (ECM) including type II collagen and sGAG were achieved in the engineered cartilage constructs. The repaired tissues possessed an average compressive modulus of 1.58MPa. For comparison, the defects without repair treatments still showed defects with fibrous tissues. These results demonstrated that PHBHHx is a useful material for cartilage tissue engineering.