Development of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers for skin tissue engineering: effects of topography, mechanical, and chemical stimuli

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, was electrospun to form defect-free fibers with high surface-area-to-volume ratio for skin regeneration. Several parameters such as solvent ratio, polymer concentration, applied voltage, flow rate, and tip-to-target distance were optimized to achieve defect-free morphology. The average diameter of the PHBV fibers was 724 ± 91 nm. PHBV was also solvent-cast to form 2-D films, and its mechanical properties, porosity, and degradation rates were compared with PHBV fibers. Our results demonstrate that PHBV fibers exhibited higher porosity, increased ductility, and faster degradation rate when compared with PHBV 2-D films (p < 0.05). In vitro studies with PHBV fibers and 2-D films were carried out to evaluate the adhesion, viability, proliferation, and gene expression of human skin fibroblasts. Cells adhered and proliferated on both PHBV fibers and 2-D films. However, the proliferation of cells on the surface of PHBV fibers was comparable to tissue culture polystyrene (TCPS, control) (p > 0.05). The gene expression of collagen I and elastin was significantly up-regulated when compared with TCPS control, whereas collagen III was down-regulated on PHBV fibers and 2-D film after 14 days in culture. The less ductile PHBV 2-D films showed higher levels of elastin expression. Furthermore, the PHBV fibers in the presence and absence of an angiogenesis factor (R-Spondin 1) were evaluated for their wound healing capacity in a rat model. The wound contracture in R-Spondin-1-loaded PHBV fibers was found to be significantly higher when compared with PHBV fibers alone after 7 days (p < 0.05). Furthermore, the presence of fibers promoted an increase in collagen and aided re-epithelialization. Thus our results demonstrate that the topography and mechanical and chemical stimuli have a pronounced influence on the cell proliferation, gene expression, and wound healing.     

Fabrication,microstructure characterization,and degradation performance of electrospun mats based on poly(3-hydroxybutyrate-co-3 hydroxyvalerate)/polyethylene glycol blend for potential tissue engineering

There have been strong demands for nanofibrous scaffolds fabricated by electrospinning for various fields due to their various advantages. Electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) fibre mats were prepared. The effects of processing variables as well as the inclusion of poly(ethylene glycol) (PEG) on the morphologies of generated fibres were investigated using Fourier-transform infrared spectroscopy and scanning electron microscopy. The average fibrous diameter was monitored in the range 400–3000 nm relying on the total content of PEG. The fluorescence cell imaging of electrospun mats was also explored. The results of cell viability demonstrated that skin fibroblast BJ-1 cells showed different adhesions and growth rates for the three kinds of PHBV fibres. Electrospun PHBV mats with low amount of PEG offer a high-quality medium for cell growth. Therefore, those mats exhibited high potential for soft tissue engineering, in particular wound healing.     

壳聚糖/ 魔芋葡甘露聚糖复合膜体内外促创面愈合研究

目的:制备壳聚糖/魔芋葡甘露聚糖复合膜,研究其促创面愈合作用。方法:壳聚糖溶液和魔芋葡甘露聚糖溶液混合后冷冻干燥制成复合膜。扫描电镜观察膜的形态和孔径,并研究比较膜的吸水率,水蒸气透过率,拉伸强度,断裂伸长率和体外降解率。建立大鼠皮肤损伤模型,敷以复合膜治疗,比较创面愈合率,观察创面组织染色结果,评价复合膜的促创面愈合作用。结果:壳聚糖/魔芋葡甘露聚糖复合膜具有三维网状结构,壳聚糖复合魔芋葡甘露聚糖后,膜的吸水率、拉伸强度和断裂伸长率提高,体外降解加速,水蒸气透过率改善。愈合实验表明壳聚糖/魔芋葡甘露聚糖膜具有促进创面愈合作用。结论:壳聚糖/魔芋葡甘露聚糖复合膜制备工艺简单,能有效促进创面愈合,具有成为创伤敷料的潜力。     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.     

In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.     

In vitro biocompatibility of schwann cells on surfaces of biocompatible polymeric electrospun fibrous and solution-cast film scaffolds

The in vitro responses of Schwann cells (RT4-D6P2T, a schwannoma cell line derived from a chemically induced rat peripheral neurotumor) on various types of electrospun fibrous scaffolds of some commercially available biocompatible and biodegradable polymers, i.e., poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polycaprolactone (PCL), poly(l-lactic acid) (PLLA), and chitosan (CS), were reported in comparison with those of the cells on corresponding solution-cast film scaffolds as well as on a tissue-culture polystyrene plate (TCPS), used as the positive control. At 24 h after cell seeding, the viability of the attached cells on the various substrates could be ranked as follows: PCL film > TCPS > PCL fibrous > PLLA fibrous > PHBV film > CS fibrous approximately CS film approximately PLLA film > PHB film > PHBV fibrous > PHB fibrous. At day 3 of cell culture, the viability of the proliferated cells on the various substrates could be ranked as follows: TCPS > PHBV film > PLLA film > PCL film > PLLA fibrous > PHB film approximately PCL fibrous > CS fibrous > CS film > PHB fibrous > PHBV fibrous. At approximately 8 h after cell seeding, the cells on the flat surfaces of all of the film scaffolds and that of the PCL nanofibrous scaffold appeared in their characteristic spindle shape, while those on the surfaces of the PHB, PHBV, and PLLA macrofibrous scaffolds also appeared in their characteristic spindle shape, but with the cells being able to penetrate to the inner side of the scaffolds.     

Adipose tissue‐derived mesenchymal stem cells and keratinocytes co‐culture on gelatin/chitosan/β‐glycerol phosphate nanoscaffold in skin regeneration

Using cell‐based engineered skin is an emerging strategy for treating difficult‐to‐heal wounds. To date, much endeavor has been devoted to the fabrication of appropriate scaffolds with suitable biomechanical properties to support cell viability and growth in the microenvironment of a wound. The aim of this research was to assess the impact of adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) and keratinocytes on gelatin/chitosan/β‐glycerol phosphate (GCGP) nanoscaffold in full‐thickness excisional skin wound healing of rats. For this purpose, AD‐MSCs and keratinocytes were isolated from rats and GCGP nanoscaffolds were electrospun. Through an in vivo study, the percentage of wound closure was assessed on days 7, 14, and 21 after wound induction. Samples were taken from the wound sites in order to evaluate the density of collagen fibers and vessels at 7 and 14 days. Moreover, sampling was done on days 7 and 14 from wound sites to assess the density of collagen fibers and vessels. The wound closure rate was significantly increased in the keratinocytes‐AD‐MSCs‐scaffold (KMS) group compared with other groups. The expressions of vascular endothelial growth factor, collagen type 1, and CD34 were also significantly higher in the KMS group compared with the other groups. These results suggest that the combination of AD‐MSCs and keratinocytes seeded onto GCGP nanoscaffold provides a promising treatment for wound healing.     

Improvement of PHBV Scaffolds with Bioglass for Cartilage Tissue Engineering

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) have proven to be possible matrices for the three-dimensional growth of chondrocyte cultures. However, the engineered cartilage grown on these PHBV scaffolds is currently unsatisfactory for clinical applications due to PHBV’s poor hydrophilicity, resulting in inadequate thickness and poor biomechanical properties of the engineered cartilage. It has been reported that the incorporation of Bioglass (BG) into PHBV can improve the hydrophilicity of the composites. In this study, we compared the effects of PHBV scaffolds and PHBV/BG composite scaffolds on the properties of engineered cartilage in vivo. Rabbit articular chondrocytes were seeded into PHBV scaffolds and PHBV/BG scaffolds. Short-term in vitro culture followed by long-term in vivo transplantation was performed to evaluate the difference in cartilage regeneration between the cartilage layers grown on PHBV and PHBV/BG scaffolds. The results show that the incorporation of BG into PHBV efficiently improved both the hydrophilicity of the composites and the percentage of adhered cells and promoted cell migration into the inner part the constructs. With prolonged incubation time in vivo, the chondrocyte-scaffold constructs in the PHBV/BG group formed thicker cartilage-like tissue with better biomechanical properties and a higher cartilage matrix content than the constructs in the PHBV/BG group. These results indicate that PHBV/BG scaffolds can be used to prepare better engineered cartilage than pure PHBV.     

In situ forming and rutin-releasing chitosan hydrogels as injectable dressings for dermal wound healing

An in situ gel-forming system composed of rutin- and tyramine-conjugated chitosan derivatives, horseradish peroxidase (HRP), and hydrogen peroxide (H(2)O(2)) was prepared and applied to dermal wound repair. Rutin was employed to enhance production and accumulation of extracellular matrix in the healing process. In vitro study demonstrates that released rutin significantly enhanced cell proliferation as compared with media without rutin. In vivo wound healing study was performed by injecting hydrogels on rat dorsal wounds with a diameter of 8 mm for 14 days. Histological results demonstrated that rutin-conjugated hydrogel exhibited enhancement of wound healing as compared with treatments with PBS, hydrogel without rutin, and a commercialized wound dressing (Duoderm). More specifically, rutin-conjugated hydrogels induced better defined formation of neo-epithelium and thicker granulation, which is closer to the original epithelial tissue. As a result, this study suggests that the in situ gel-forming system can be a promising injectable gel-type wound dressing.     

Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3(+)(/)(+)) and Tff3-deficient (Tff3(-)(/)(-)) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3(-/-) mice is significantly prolonged in comparison to Tff3(+/+) mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3(+/+) and Tff3(-/-) mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.     

Development of a chitosan nanofibrillar scaffold for skin repair and regeneration

The final goal of the present study was the development of a 3-D chitosan dressing that would shorten the healing time of skin wounds by stimulating migration, invasion, and proliferation of the relevant cutaneous resident cells. Three-dimensional chitosan nanofibrillar scaffolds produced by electrospinning were compared with evaporated films and freeze-dried sponges for their biological properties. The nanofibrillar structure strongly improved cell adhesion and proliferation in vitro. When implanted in mice, the nanofibrillar scaffold was colonized by mesenchymal cells and blood vessels. Accumulation of collagen fibrils was also observed. In contrast, sponges induced a foreign body granuloma. When used as a dressing covering full-thickness skin wounds in mice, chitosan nanofibrils induced a faster regeneration of both the epidermis and dermis compartments. Altogether our data illustrate the critical importance of the nanofibrillar structure of chitosan devices for their full biocompatibility and demonstrate the significant beneficial effect of chitosan as a wound-healing biomaterial.     

Characterization of a new degradable polymer scaffold for regeneration of the dermis: In vitro and in vivo human studies

Full thickness skin wounds in humans heal with scars, but without regeneration of the dermis. A degradable poly(urethane urea) scaffold (PUUR), Artelon® is already used to reinforce soft tissues in orthopaedics, and for treatment of osteoarthritis of the hand, wrist and foot. In this paper we have done in vitro experiments followed by in vivo studies to find out whether the PUUR is biocompatible and usable as a template for dermal regeneration. Human dermal fibroblasts were cultured on discs of PUUR, with different macrostructures (fibrous and porous). They adhered to and migrated into the scaffolds, and produced collagen. The porous scaffold was judged more suitable for clinical applications and 4 mm Ø, 2 mm-thick discs of porous scaffold (12% w/w or 9% w/w polymer solution) were inserted intradermally in four healthy human volunteers. The implants were well tolerated and increasing ingrowth of fibroblasts was seen over time in all subjects. The fibroblasts stained immunohistochemically for procollagen and von Willebrand factor, indicating neocollagenesis and angiogenesis within the scaffolds. The PUUR scaffold may be a suitable material to use as a template for dermal regeneration.Key words: dermal regeneration, tissue engineering, polymer scaffold, wound healing, in vitro, in vivo, guided tissue regeneration, human, burns     

Anti-biofouling 3D porous systems: the blend effect of oxazoline-based oligomers on chitosan scaffolds

The production, characterization and anti-biofouling activity of 3D porous scaffolds combining different blends of chitosan and oxazoline-based antimicrobial oligomers is reported. The incorporation of ammonium quaternized oligo(2-oxazoline)s into the composition of the scaffold enhances the stability of the chitosan scaffold under physiological conditions as well as its ability to repel protein adsorption. The blended scaffolds showed mean pore sizes in the range of 18–32?μm, a good pore interconnectivity and high porosity, as well as a large surface area, ultimate key features for anti-biofouling applications. Bovine serum albumin (BSA) adhesion profiles showed that the composition of the scaffolds plays a critical role in the chitosan–oligooxazoline system. Oligobisoxazoline-enriched scaffolds (20%?w/w, CB8020) decreased protein adsorption (BSA) by up to 70%. Moreover, 1?mg of CB8020 was able to kill 99.9% of Escherichia coli cells upon contact, demonstrating its potential as promising material for production of tailored non-fouling 3D structures to be used in the construction of novel devices with applications in the biomedical field and water treatment processes.     

Biopolymer gel matrix as acellular scaffold for enhanced dermal tissue regeneration

Biological grafts have drawbacks such as donor scarcity, disease transmission, tissue infection, while the scaffolds of either collagen or chitosan fabrics fail to become part of the tissue at the wound site, though they favor the formation of connective tissue matrix. This study developed a novel composite consisting of the combination of atelocollagen and chitosan in order to provide a biodegradable molecular matrix in gel form as a biomimetic surface for cell attachment, to promote the wound healing in excision wounds. We found that the topical application of biopolymer composite on the wound promoted cell proliferation, migration and collagen deposition overtime. The enhanced cellular activity in the collagen-chitosan treated wound tissue was also assed by increased levels of Platelet derived growth factor (PDGF) and Nerve growth factor (NGF) associated with elevated levels of antioxidants and decreased level of lipid peroxidation. The acellular matrix-like topical application material is designed to guide the eventual re-establishment of an anatomically normal skin. The results of this study demonstrate the feasibility of multi-cell regeneration on a molecular system that mimics tissue engineering in vivo.     

Electrospun Scaffold of Collagen and Polycaprolactone Containing ZnO Quantum Dots for Skin Wound Regeneration

Nanofibers(NFs)have been widely used in tissue engineering such as wound healing.In this work,the antibacterial ZnO quantum dots(ZnO QDs)have been incorporated into the biocompatible poly(ε-caprolactone)/collagen(PCL/Col)fibrous scaffolds for wound healing.The as-fabricated PCL-Col/ZnO fibrous scaffolds exhibited good swelling,antibacterial activity,and biodegradation behaviors,which were beneficial for the applications as a wound dressing.Moreover,the PCL-Col/ZnO fibrous scaffolds showed excellent cytocompatibility for promoting cell proliferation.The resultant PCL-Col/ZnO fibrous scaffolds containing vascular endothelial growth factor(VEGF)also exhibited promoted wound-healing effect through promoting expression of transforming growth factor-β(TGF-β)and the vascular factor(CD31)in tissues in the early stages of wound healing.This new electrospun fibrous scaffolds with wound-healing promotion and antibacterial property should be convenient for treating wound healing.     

Retracted: Paeoniflorin promotes angiogenesis and tissue regeneration in a full-thickness cutaneous wound model through the PI3K/AKT pathway

The treatment of wounds remains a clinical challenge because of poor angiogenesis under the wound bed, and increasingly, the patients' need for functional and aesthetically pleasing scars. For the wound healing process, new blood vessels which can deliver nutrients and oxygen to the wound area are necessary. In this study, we investigated the pro-angiogenesis ability and mechanism in wound healing of paeoniflorin (PF), which is a traditional Chinese medicine. In our in vitro results, the ability for proliferation, migration and in vitro angiogenesis in human umbilical vein endothelial cells was promoted by coculturing with PF (1.25–5 μM). Meanwhile, molecular docking studies revealed that PF has excellent binding abilities to phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT), and consistent with our western blot results, that PF suppressed PI3K and AKT phosphorylation. Furthermore, to investigate the healing effect of PF in vivo, we constructed a full-thickness cutaneous wound model in rats. PF stimulated the cellular proliferation status, collagen matrix deposition and remodeling processes in vitro and new blood vessel formation at the wound bed resulting in efficient wound healing after intragastric administration of 10 mg·kg⁻¹·day⁻¹ in vivo. Overall, PF performed the pro-angiogenetic effect in vitro and accelerating wound healing in vivo. In summary, the capacity for angiogenesis in endothelial cells could be enhanced by PF treatment via the PI3K/AKT pathway in vitro and could accelerate the wound healing process in vivo through collagen deposition and angiogenesis in regenerated tissue. This study provides evidence that application of PF represents a novel therapeutic approach for the treatment of cutaneous wounds.     

Engineering a bilayered hydrogel to control ASC differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM). Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.     

Fabrication,Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm⁻¹ can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.     

Poly(3-hydroxubutyrate-co-4-hydroxubutyrate)/bacterial cellulose composite porous scaffold: Preparation, characterization and biocompatibility evaluation

Bio-composite scaffolds were prepared by freeze-drying using poly(3-hydroxubutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) and bacterial cellulose (BC) as raw materials and trifluoroacetic acid (TFA) as co-solvent. The characteristics of the composite scaffold were investigated by field emission scanning electron microscopy (FESEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), water contact angle measurement and tensile testing. Preliminary biodegradation test was performed for P(3HB-co-4HB) and P(3HB-co-4HB)/BC composite scaffold in buffer solution and enzyme solution. The biocompatibility of the composite scaffold was preliminarily evaluated by cell adhesion studies using Chinese Hamster Lung (CHL) fibroblast cells. The cells incubated with composite scaffold for 48 h were capable of forming cell adhesion and proliferation, which showed better biocompatibility than pure P(3HB-co-4HB) scaffold. Thus, the prepared P(3HB-co-4HB)/BC composite scaffold was bioactive and may be suitable for cell adhesion/attachment suggesting that these scaffolds can be used for wound dressing or tissue-engineering scaffolds.     

Preparation of 3D fibrin scaffolds for stem cell culture applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach.