Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal prolifer... more Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal proliferation of myeloid blasts. Despite the progress that has been made in the treatment of various malignant hematopoietic diseases, the effective treatment of AML remains very challenging. Differentiation therapy has emerged as a promising approach for leukemia treatment, and new and effective chemical agents to trigger the differentiation of AML cells, especially drug-resistant cells, are urgently required. Herein, the natural product jungermannenone C, a tetracyclic diterpenoid isolated from liverworts, is reported to induce cell differentiation in AML cells. Interestingly, the unnatural enantiomer of jungermannenone C (1) was found to be more potent than jungermannenone C in inducing cell differentiation. Furthermore, compound 1 targets peroxiredoxins I and II by selectively binding to the conserved cysteine residues and leads to cellular reactive oxygen species accumulation. Accordingly, ent-jungermannenone C (1) shows potential for further investigation as an effective differentiation therapy against AML.
Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx) has superior mechanical property and bioco... more Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx) has superior mechanical property and biocompatibility that may enable it to meet cardiovascular tissue engineering applications. We developed hybrid materials based on decellularized xenogenic vascular scaffolds that were coated with PHBHHx to investigate the intravascular biocompatibility. The hybrid patches were implanted in the decellularized xenogenic vascular scaffolds of the rabbit abdominal aorta at 1, 4, and 12 weeks after surgery. In conclusion, PHBHHx showed remarkable intravascular biocompatibility and will benefit endothelization, which will be a useful candidate for lumen of cardiovascular tissue engineering.
As a product of a multi-step enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in... more As a product of a multi-step enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by over-expression of PHB synthesis pathway from native producer involving three genes phbC, phbA and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here we reported a semi-rational approach for high-efficient PHB pathway optimization in E. coli based on phbCAB operon cloned from native producer Ralstonia entropha (R. entropha). Rational designed Ribosomal Binding Sites (RBS) libraries with defined strengths for each of the three genes, were constructed based on high or low copy number plasmids in a one-pot reaction by an Oligo-Linker Mediated Assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high throughput screening and detailed in-depth analysis. Applying this approach, s...
Halomonas strain TD01, a newly identified halophilic bacterium, has proven to be a promising low-... more Halomonas strain TD01, a newly identified halophilic bacterium, has proven to be a promising low-cost host for the production of chemicals. However, genetic manipulation in Halomonas sp. is still difficult due to the lack of well-characterized and tunable expression systems. In this study, a systematic, efficient method was exploited to construct both a constitutive promoter library and inducible promoters. Porin, a highly expressed protein in Halomonas TD01, was first identified from the Halomonas TD01 proteome. Subsequent study of the intergenic region upstream of porin led to the identification of a core promoter region, including -10 and -35 elements. By randomizing the sequence between the -35 and -10 elements, a constitutive promoter library was obtained with 310-fold variation in transcriptional activity; an inducible promoter with a >200-fold induction was built by integrating the LacI operator into the core promoter region. As two complementary expression systems, the co...
The first AFOB special issue of Biotechnology Journal is edited by Prof. Tai Hyun Park and Prof. ... more The first AFOB special issue of Biotechnology Journal is edited by Prof. Tai Hyun Park and Prof. George G. Q. Chen. The eleven articles are representative of the diverse nature of biotechnology today, covering topics such as microfluidic devices, high-throughput analysis, biosensors, bio-imaging, tissue engineering, vaccination, gene delivery, gene expression, and cell-free protein synthesis.
Journal of biomedical materials research. Part A, 2015
Human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs) have been widely used in tis... more Human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs) have been widely used in tissue engineering. The aim of this study is to evaluate the ability of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds coated with polyhydroxyalkanoate binding protein fused with arginyl-glycyl-aspartic acid (PhaP-RGD) to promote the proliferation and chondrogenic differentiation of hUC-MSCs seeded on them. The PhaP-RGD fusion protein was expressed by Escherichia coli. PHBHHx films were coated with PhaP-RGD fusion protein and the physiochemical properties were examined. hUC-MSCs were seeded on PHBHHx films with or without PhaP-RGD precoating and tested for changes in morphology, viability, and chondrogenic differentiation. We found that PhaP-RGD-coated PHBHHx films had similar surface morphology to uncoated PHBHHx. The water contact angle of the coated PHBHHx surface was lower than that of the uncoated surface (10.63° vs. 98.69°). At 7 and 14 days after seeding, the PhaP-...
The ability to control the size and quality of nanoparticles (NPs) during production is critical ... more The ability to control the size and quality of nanoparticles (NPs) during production is critical for their success as a commercial product for clinical applications. Here, we employed a statistical design of experiment approach to identify the key process variables affecting the size of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) NPs during production via the solvent evaporation method. The number of sonication cycles had a standardzed effect on NP size of 55, with sonication power at 25, and PHBHHx concentration at 27 with a combination of these variables having a lower yet significant effect on NP size (p < 0.05). The PHBHHx NPs were stable for at least 7 days with an average polydispersity index of 0.18, a zeta potential of -10 to -40 mV, and an encapsulation efficiency of 63.5 ± 2%. These data were utilized to produce a prediction graph whereby particles could be produced with sizes ranging from 90 to 205 nm with a low mean curve prediction error of 1.96% for Haper...
Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past ... more Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past many years. However, PHA still have only a very limited market. Because of the availability of large amount of shale gas, petroleum will not raise dramatically in price, this situation makes PHA less competitive compared with low cost petroleum based plastics. Therefore, two strategies have been adopted to meet this challenge: first, the development of a super PHA production strain combined with advanced fermentation processes to produce PHA at a low cost; second, the construction of functional PHA production strains with technology to control the precise structures of PHA molecules, this will allow the resulting PHA with high value added applications. The recent systems and synthetic biology approaches allow the above two strategies to be implemented. In the not so distant future, the new technology will allow PHA to be produced with a competitive price compared with petroleum-based pla...
Microbial fermentation is the key to industrial biotechnology. Most fermentation processes are se... more Microbial fermentation is the key to industrial biotechnology. Most fermentation processes are sensitive to microbial contamination and require an energy intensive sterilization process. The majority of microbial fermentations can only be conducted over a short period of time in a batch or fed-batch culture, further increasing energy consumption and process complexity, and these factors contribute to the high costs of bio-products. In an effort to make bio-products more economically competitive, increased attention has been paid to developing open (unsterile) and continuous processes. If well conducted, continuous fermentation processes will lead to the reduced cost of industrial bio-products. To achieve cost-efficient open and continuous fermentations, the feeding of raw materials and the removal of products must be conducted in a continuous manner without the risk of contamination, even under 'open' conditions. Factors such as the stability of the biological system as a wh...
Industrial biotechnology has not developed as fast as expected due to some challenges including t... more Industrial biotechnology has not developed as fast as expected due to some challenges including the emergences of alternative energy sources, especially shale gas, natural gas hydrate (or gas hydrate) and sand oil et al. The weaknesses of microbial or enzymatic processes compared with the chemical processing also make industrial biotech products less competitive with the chemical ones. However, many opportunities are still there if industrial biotech processes can be as similar as the chemical ones. Taking advantages of the molecular biology and synthetic biology methods as well as changing process patterns, we can develop bioprocesses as competitive as chemical ones, these including the minimized cells, open and continuous fermentation processes et al.
Poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3‐hydroxybuty... more Poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3‐hydroxybutyrate (3HB) and 3‐hydroxyhexanoate (3HHx), can be synthesized by Aeromonas hydrophila strain 4AK4 using long chain fatty acids as the carbon source. The wild type A. hydrophila 4AK4 accumulated PHBHHx consisting of 12‐15 mol% 3HHx regardless of growth conditions. When phbA, phbB and vgb genes encoding β‐ketothiolase, acetoacetyl‐CoA reductase and vitreoscilla hemoglobin, respectively, were introduced together into A. hydrophila 4AK4, the recombinant strain grew to over 20 g/L cell dry weight (CDW) after 48 h of shake flask cultivation in co‐substrates of dodecanoate and gluconate (weight ratio 1:1), and the CDW contained 50% PHBHHx consisting of 9 mol% 3HHx. Under similar conditions, the wild type strain produced only 12 g/L CDW containing 32% PHBHHx with 15 mol% 3HHx. In comparison, recombinant harboring phbA and phbB produced 35% PHBHHx with 9 mol% 3HHx in 15 g/L CDW under the same cond...
Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2013
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible and bioresorbable copol... more Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible and bioresorbable copolymer that has generated research interest as a bone scaffold material. However, its brittleness and degradation characteristics can be improved upon. We hypothesized that blending with medical-grade polycaprolactone (PCL) can improve degradation and mechanical characteristics. Here, we report the development of solvent-blended PHBHHx/PCL for application as a potential biomaterial for tissue engineering. Enhanced yield strength, yield strain and Young&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s modulus occurred at 30/70 blend when compared with PHBHHx and PCL. Polarized light microscopy demonstrated PHBHHx and PCL to exist as morphologically and optically distinct phases and, together with thermal analyses, revealed immiscibility. Hydrophilicity improved with the addition of PCL. Accelerated hydrolytic studies suggested predictable behavior of PHBHHx/PCL. Notably, 30/70 blend exhibited similar degradation behavior to PCL in terms of changes in crystallinity, molecular weight, morphology, and mass loss. Finally, human fetal mesenchymal stem cells (hfMSCs) were evaluated on PHBHHx/PCL using live/dead assay and results suggested encouraging hfMSC adhesion and proliferative capacity, with near-confluence occurring in PHBHHx and 30/70 blend after 5 days. Taken together, these are encouraging results for the further development of PHBHHx/PCL as a potential biomaterial for tissue engineering.
Journal of Biomedical Materials Research Part A, 2011
In this study, a novel tissue engineering scaffold material of electrospun silk fibroin/nano‐hydr... more In this study, a novel tissue engineering scaffold material of electrospun silk fibroin/nano‐hydroxyapatite (nHA) biocomposite was prepared by means of an effective calcium and phosphate (Ca‐P) alternate soaking method. nHA was successfully produced on regenerated silk fibroin nanofiber as a substrate within several minutes without any pretreatments. The morphologies of both nonmineralized and mineralized nanofibers were analyzed using a field‐emission scanning electron microscopy (FESEM). The crystallographic phases of the nHA were analyzed using X‐ray diffraction (XRD). Fourier transform infrared (FTIR) spectrophotometer and thermogravimetry analyses (TGA) were employed to determine the type of functional groups and the amount of nHA presenting in the silk/nHA biocomposite nanofibers, respectively. The osteoblastic activities of this novel nanofibrous biocomposite scaffold were also investigated by employing osteoblastic‐like MC3T3‐E1 cell line. The cell functionality such as alka...
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and PHBHHx—hydroxyapatite (HAP) composite ... more 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.
Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalk... more Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalkanoate synthase gene (phaC Ac ) and (R)-enoyl-CoA hydratase gene (phaJ Ac ) from Aeromonas caviae. The expression plasmids were introduced into E. coli JM109, DH5K and XL1-blue, respectively. Compared with the strains harboring only phaC Ac and phaJ Ac , all recombinant E. coli strains harboring yafH, phaC Ac and phaJ Ac accumulated at least four times more poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Cell dry weights produced by all recombinants containing yafH were also considerably higher than that without yafH. The addition of acrylic acid which serves as inhibitor for L-oxidation and may lead to more precursor supply for PHA synthesis did not result in improved PHBHHx production compared with that of the overexpression of yafH. It appeared that the overexpression of acyl-CoA dehydrogenase gene (yafH) enhanced the supply of enoyl-CoA which is the substrate of (R)-enoyl-CoA hydratase. With the enhanced precursor supply, the recombinants accumulated more PHBHHx.
Many bacteria have been found to produce various polyhydroxyalkanoates (PHA) biopolyesters. In ma... more Many bacteria have been found to produce various polyhydroxyalkanoates (PHA) biopolyesters. In many cases, it is not easy to control the structures of PHA including homopolymers, random copolymers and block copolymers as well as ratios of monomers in the copolymers. It has become possible to engineer bacteria for controllable synthesis of PHA with the desirable structures by creating new PHA synthesis pathways. Remarkably, the weakening of b-oxidation cycle in Pseudomonas putida and Pseudomonas entomophila led to controllable synthesis of all kinds of PHA structures including monomer ratios in random and/or block copolymers when fatty acids are used as PHA precursors. Introduction of functional groups into PHA polymer chains in predefined proportions has become a reality provided fatty acids containing the functional groups are taken up by the bacteria for PHA synthesis. This allows the formation of functional PHA for further grafting. The PHA diversity is further widened by the endless possibility of controllable homopolymerization, random copolymerization, block copolymerization and grafting on functional PHA site chains.
Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth cond... more Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/ regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future.
Polyhydroxyalkanoates, abbreviated as PHA, have been studied for medical applications due to thei... more Polyhydroxyalkanoates, abbreviated as PHA, have been studied for medical applications due to their suitable mechanical properties, blood and tissue tolerance and in vivo biodegradability. As a new member of PHA family, terpolyester of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate, abbreviated as PHBVHHx, was compared with polylactic acid (PLA), copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) for their respective functions leading to differentiation of human bone marrow mesenchymal stem cell (hBMSC) into nerve cells. Results indicated that 3D scaffolds promoted the differentiation of hBMSC into nerve cells more intensively compared with 2D films. Smaller pore sizes of scaffolds increased differentiation of hBMSC into nerve cells, whereas decreased cell proliferation. PHBVHHx scaffolds with pore sizes of 30-60 mm could be used in nerve tissue engineering for treatment of nerve injury. The above results were supported by scanning electron microscope (SEM) and confocal microscopy observation on attachment and growth of hBMSCs on PLA, PHBHHx and PHBVHHx, and by CCK-8 evaluation of cell proliferation. In addition, expressions of nerve markers nestin, GFAP and b-III tubulin of nerve cells differentiated from hBMSC grown in PHBVHHx scaffolds were confirmed by real-time PCR.
Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisti... more Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the β-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.
Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal prolifer... more Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal proliferation of myeloid blasts. Despite the progress that has been made in the treatment of various malignant hematopoietic diseases, the effective treatment of AML remains very challenging. Differentiation therapy has emerged as a promising approach for leukemia treatment, and new and effective chemical agents to trigger the differentiation of AML cells, especially drug-resistant cells, are urgently required. Herein, the natural product jungermannenone C, a tetracyclic diterpenoid isolated from liverworts, is reported to induce cell differentiation in AML cells. Interestingly, the unnatural enantiomer of jungermannenone C (1) was found to be more potent than jungermannenone C in inducing cell differentiation. Furthermore, compound 1 targets peroxiredoxins I and II by selectively binding to the conserved cysteine residues and leads to cellular reactive oxygen species accumulation. Accordingly, ent-jungermannenone C (1) shows potential for further investigation as an effective differentiation therapy against AML.
Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx) has superior mechanical property and bioco... more Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx) has superior mechanical property and biocompatibility that may enable it to meet cardiovascular tissue engineering applications. We developed hybrid materials based on decellularized xenogenic vascular scaffolds that were coated with PHBHHx to investigate the intravascular biocompatibility. The hybrid patches were implanted in the decellularized xenogenic vascular scaffolds of the rabbit abdominal aorta at 1, 4, and 12 weeks after surgery. In conclusion, PHBHHx showed remarkable intravascular biocompatibility and will benefit endothelization, which will be a useful candidate for lumen of cardiovascular tissue engineering.
As a product of a multi-step enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in... more As a product of a multi-step enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by over-expression of PHB synthesis pathway from native producer involving three genes phbC, phbA and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here we reported a semi-rational approach for high-efficient PHB pathway optimization in E. coli based on phbCAB operon cloned from native producer Ralstonia entropha (R. entropha). Rational designed Ribosomal Binding Sites (RBS) libraries with defined strengths for each of the three genes, were constructed based on high or low copy number plasmids in a one-pot reaction by an Oligo-Linker Mediated Assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high throughput screening and detailed in-depth analysis. Applying this approach, s...
Halomonas strain TD01, a newly identified halophilic bacterium, has proven to be a promising low-... more Halomonas strain TD01, a newly identified halophilic bacterium, has proven to be a promising low-cost host for the production of chemicals. However, genetic manipulation in Halomonas sp. is still difficult due to the lack of well-characterized and tunable expression systems. In this study, a systematic, efficient method was exploited to construct both a constitutive promoter library and inducible promoters. Porin, a highly expressed protein in Halomonas TD01, was first identified from the Halomonas TD01 proteome. Subsequent study of the intergenic region upstream of porin led to the identification of a core promoter region, including -10 and -35 elements. By randomizing the sequence between the -35 and -10 elements, a constitutive promoter library was obtained with 310-fold variation in transcriptional activity; an inducible promoter with a >200-fold induction was built by integrating the LacI operator into the core promoter region. As two complementary expression systems, the co...
The first AFOB special issue of Biotechnology Journal is edited by Prof. Tai Hyun Park and Prof. ... more The first AFOB special issue of Biotechnology Journal is edited by Prof. Tai Hyun Park and Prof. George G. Q. Chen. The eleven articles are representative of the diverse nature of biotechnology today, covering topics such as microfluidic devices, high-throughput analysis, biosensors, bio-imaging, tissue engineering, vaccination, gene delivery, gene expression, and cell-free protein synthesis.
Journal of biomedical materials research. Part A, 2015
Human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs) have been widely used in tis... more Human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs) have been widely used in tissue engineering. The aim of this study is to evaluate the ability of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds coated with polyhydroxyalkanoate binding protein fused with arginyl-glycyl-aspartic acid (PhaP-RGD) to promote the proliferation and chondrogenic differentiation of hUC-MSCs seeded on them. The PhaP-RGD fusion protein was expressed by Escherichia coli. PHBHHx films were coated with PhaP-RGD fusion protein and the physiochemical properties were examined. hUC-MSCs were seeded on PHBHHx films with or without PhaP-RGD precoating and tested for changes in morphology, viability, and chondrogenic differentiation. We found that PhaP-RGD-coated PHBHHx films had similar surface morphology to uncoated PHBHHx. The water contact angle of the coated PHBHHx surface was lower than that of the uncoated surface (10.63° vs. 98.69°). At 7 and 14 days after seeding, the PhaP-...
The ability to control the size and quality of nanoparticles (NPs) during production is critical ... more The ability to control the size and quality of nanoparticles (NPs) during production is critical for their success as a commercial product for clinical applications. Here, we employed a statistical design of experiment approach to identify the key process variables affecting the size of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) NPs during production via the solvent evaporation method. The number of sonication cycles had a standardzed effect on NP size of 55, with sonication power at 25, and PHBHHx concentration at 27 with a combination of these variables having a lower yet significant effect on NP size (p < 0.05). The PHBHHx NPs were stable for at least 7 days with an average polydispersity index of 0.18, a zeta potential of -10 to -40 mV, and an encapsulation efficiency of 63.5 ± 2%. These data were utilized to produce a prediction graph whereby particles could be produced with sizes ranging from 90 to 205 nm with a low mean curve prediction error of 1.96% for Haper...
Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past ... more Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past many years. However, PHA still have only a very limited market. Because of the availability of large amount of shale gas, petroleum will not raise dramatically in price, this situation makes PHA less competitive compared with low cost petroleum based plastics. Therefore, two strategies have been adopted to meet this challenge: first, the development of a super PHA production strain combined with advanced fermentation processes to produce PHA at a low cost; second, the construction of functional PHA production strains with technology to control the precise structures of PHA molecules, this will allow the resulting PHA with high value added applications. The recent systems and synthetic biology approaches allow the above two strategies to be implemented. In the not so distant future, the new technology will allow PHA to be produced with a competitive price compared with petroleum-based pla...
Microbial fermentation is the key to industrial biotechnology. Most fermentation processes are se... more Microbial fermentation is the key to industrial biotechnology. Most fermentation processes are sensitive to microbial contamination and require an energy intensive sterilization process. The majority of microbial fermentations can only be conducted over a short period of time in a batch or fed-batch culture, further increasing energy consumption and process complexity, and these factors contribute to the high costs of bio-products. In an effort to make bio-products more economically competitive, increased attention has been paid to developing open (unsterile) and continuous processes. If well conducted, continuous fermentation processes will lead to the reduced cost of industrial bio-products. To achieve cost-efficient open and continuous fermentations, the feeding of raw materials and the removal of products must be conducted in a continuous manner without the risk of contamination, even under 'open' conditions. Factors such as the stability of the biological system as a wh...
Industrial biotechnology has not developed as fast as expected due to some challenges including t... more Industrial biotechnology has not developed as fast as expected due to some challenges including the emergences of alternative energy sources, especially shale gas, natural gas hydrate (or gas hydrate) and sand oil et al. The weaknesses of microbial or enzymatic processes compared with the chemical processing also make industrial biotech products less competitive with the chemical ones. However, many opportunities are still there if industrial biotech processes can be as similar as the chemical ones. Taking advantages of the molecular biology and synthetic biology methods as well as changing process patterns, we can develop bioprocesses as competitive as chemical ones, these including the minimized cells, open and continuous fermentation processes et al.
Poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3‐hydroxybuty... more Poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3‐hydroxybutyrate (3HB) and 3‐hydroxyhexanoate (3HHx), can be synthesized by Aeromonas hydrophila strain 4AK4 using long chain fatty acids as the carbon source. The wild type A. hydrophila 4AK4 accumulated PHBHHx consisting of 12‐15 mol% 3HHx regardless of growth conditions. When phbA, phbB and vgb genes encoding β‐ketothiolase, acetoacetyl‐CoA reductase and vitreoscilla hemoglobin, respectively, were introduced together into A. hydrophila 4AK4, the recombinant strain grew to over 20 g/L cell dry weight (CDW) after 48 h of shake flask cultivation in co‐substrates of dodecanoate and gluconate (weight ratio 1:1), and the CDW contained 50% PHBHHx consisting of 9 mol% 3HHx. Under similar conditions, the wild type strain produced only 12 g/L CDW containing 32% PHBHHx with 15 mol% 3HHx. In comparison, recombinant harboring phbA and phbB produced 35% PHBHHx with 9 mol% 3HHx in 15 g/L CDW under the same cond...
Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2013
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible and bioresorbable copol... more Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible and bioresorbable copolymer that has generated research interest as a bone scaffold material. However, its brittleness and degradation characteristics can be improved upon. We hypothesized that blending with medical-grade polycaprolactone (PCL) can improve degradation and mechanical characteristics. Here, we report the development of solvent-blended PHBHHx/PCL for application as a potential biomaterial for tissue engineering. Enhanced yield strength, yield strain and Young&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s modulus occurred at 30/70 blend when compared with PHBHHx and PCL. Polarized light microscopy demonstrated PHBHHx and PCL to exist as morphologically and optically distinct phases and, together with thermal analyses, revealed immiscibility. Hydrophilicity improved with the addition of PCL. Accelerated hydrolytic studies suggested predictable behavior of PHBHHx/PCL. Notably, 30/70 blend exhibited similar degradation behavior to PCL in terms of changes in crystallinity, molecular weight, morphology, and mass loss. Finally, human fetal mesenchymal stem cells (hfMSCs) were evaluated on PHBHHx/PCL using live/dead assay and results suggested encouraging hfMSC adhesion and proliferative capacity, with near-confluence occurring in PHBHHx and 30/70 blend after 5 days. Taken together, these are encouraging results for the further development of PHBHHx/PCL as a potential biomaterial for tissue engineering.
Journal of Biomedical Materials Research Part A, 2011
In this study, a novel tissue engineering scaffold material of electrospun silk fibroin/nano‐hydr... more In this study, a novel tissue engineering scaffold material of electrospun silk fibroin/nano‐hydroxyapatite (nHA) biocomposite was prepared by means of an effective calcium and phosphate (Ca‐P) alternate soaking method. nHA was successfully produced on regenerated silk fibroin nanofiber as a substrate within several minutes without any pretreatments. The morphologies of both nonmineralized and mineralized nanofibers were analyzed using a field‐emission scanning electron microscopy (FESEM). The crystallographic phases of the nHA were analyzed using X‐ray diffraction (XRD). Fourier transform infrared (FTIR) spectrophotometer and thermogravimetry analyses (TGA) were employed to determine the type of functional groups and the amount of nHA presenting in the silk/nHA biocomposite nanofibers, respectively. The osteoblastic activities of this novel nanofibrous biocomposite scaffold were also investigated by employing osteoblastic‐like MC3T3‐E1 cell line. The cell functionality such as alka...
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and PHBHHx—hydroxyapatite (HAP) composite ... more 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.
Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalk... more Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalkanoate synthase gene (phaC Ac ) and (R)-enoyl-CoA hydratase gene (phaJ Ac ) from Aeromonas caviae. The expression plasmids were introduced into E. coli JM109, DH5K and XL1-blue, respectively. Compared with the strains harboring only phaC Ac and phaJ Ac , all recombinant E. coli strains harboring yafH, phaC Ac and phaJ Ac accumulated at least four times more poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Cell dry weights produced by all recombinants containing yafH were also considerably higher than that without yafH. The addition of acrylic acid which serves as inhibitor for L-oxidation and may lead to more precursor supply for PHA synthesis did not result in improved PHBHHx production compared with that of the overexpression of yafH. It appeared that the overexpression of acyl-CoA dehydrogenase gene (yafH) enhanced the supply of enoyl-CoA which is the substrate of (R)-enoyl-CoA hydratase. With the enhanced precursor supply, the recombinants accumulated more PHBHHx.
Many bacteria have been found to produce various polyhydroxyalkanoates (PHA) biopolyesters. In ma... more Many bacteria have been found to produce various polyhydroxyalkanoates (PHA) biopolyesters. In many cases, it is not easy to control the structures of PHA including homopolymers, random copolymers and block copolymers as well as ratios of monomers in the copolymers. It has become possible to engineer bacteria for controllable synthesis of PHA with the desirable structures by creating new PHA synthesis pathways. Remarkably, the weakening of b-oxidation cycle in Pseudomonas putida and Pseudomonas entomophila led to controllable synthesis of all kinds of PHA structures including monomer ratios in random and/or block copolymers when fatty acids are used as PHA precursors. Introduction of functional groups into PHA polymer chains in predefined proportions has become a reality provided fatty acids containing the functional groups are taken up by the bacteria for PHA synthesis. This allows the formation of functional PHA for further grafting. The PHA diversity is further widened by the endless possibility of controllable homopolymerization, random copolymerization, block copolymerization and grafting on functional PHA site chains.
Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth cond... more Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/ regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future.
Polyhydroxyalkanoates, abbreviated as PHA, have been studied for medical applications due to thei... more Polyhydroxyalkanoates, abbreviated as PHA, have been studied for medical applications due to their suitable mechanical properties, blood and tissue tolerance and in vivo biodegradability. As a new member of PHA family, terpolyester of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate, abbreviated as PHBVHHx, was compared with polylactic acid (PLA), copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) for their respective functions leading to differentiation of human bone marrow mesenchymal stem cell (hBMSC) into nerve cells. Results indicated that 3D scaffolds promoted the differentiation of hBMSC into nerve cells more intensively compared with 2D films. Smaller pore sizes of scaffolds increased differentiation of hBMSC into nerve cells, whereas decreased cell proliferation. PHBVHHx scaffolds with pore sizes of 30-60 mm could be used in nerve tissue engineering for treatment of nerve injury. The above results were supported by scanning electron microscope (SEM) and confocal microscopy observation on attachment and growth of hBMSCs on PLA, PHBHHx and PHBVHHx, and by CCK-8 evaluation of cell proliferation. In addition, expressions of nerve markers nestin, GFAP and b-III tubulin of nerve cells differentiated from hBMSC grown in PHBVHHx scaffolds were confirmed by real-time PCR.
Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisti... more Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the β-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.
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Papers by Guo-Qiang Chen