Chun-Hsu Yao

Properties of the poly(vinyl alcohol)/chitosan blend and its effect on the culture of fibroblast in vitro

In this work, the properties of poly(vinyl alcohol) (PVA) and PVA/chitosan blended membranes were investigated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and electron spectroscopy for chemical analysis (ESCA). The SEM photographs show the PVA/chitosan blended membrane undergoes dramatic changes on the surface and bulk structure during the membrane formation. The DSC analysis shows that PVA and chitosan are not very compatible in the PVA/chitosan blended membrane, whereas the combination of two polymer chains of constitutionally different features is revealed. In addition, the surface of the PVA/chitosan blended membrane is enriched with nitrogen atoms at the ESCA analysis. These reflect the PVA membrane can be modified by blending with chitosan that in turn may affect the biocompatibility of the blended membrane. Therefore, adhesion and growth of fibroblasts on the PVA as well as PVA/chitosan blended membranes were investigated. Cell morphologies on the membranes were examined by SEM and cell viability was studied using MTT assay. It was observed that the PVA/chitosan blended membrane was more favorable for the cell culture than the pure PVA membrane. Cells cultured on the PVA/chitosan blended membrane had good spreading, cytoplasm webbing and flattening and were more compacting than on the pure PVA membrane. Consequently, the PVA/chitosan blended membrane may spatially mediate cellular response that can promote cell attachment and growth, indicating the PVA/chitosan blended membrane should be useful as a biomaterial for cell culture.

Biological effects and cytotoxicity of the composite composed by tricalcium phosphate and glutaraldehyde cross-linked gelatin.

The purpose of this study was to prepare and evaluate the feasibility and cytocompatibility of a composite (GTG) as a large defect bone substitute. The composite is combined with tricalcium phosphate ceramic particles and glutaraldehyde cross-linked gelatin. Gelatin had been reported as an adhesive and biocompatible binder that could accelerate the recovery of damaged soft tissue, but the effects of gelatin when acting on the bone tissue is not clear. Thus, it is necessary to determine if the substances released from the GTG composite can facilitate the growth of bone cells. The substances released from the GTG composites after being soaked in deionized distilled water were analyzed by gas chromatography (GC), ultraviolet and visible absorption spectroscopy (UV-VIS), and inductive-coupled plasma-atomic emission spectrometry (ICP-AES). The cytotoxicity of the GTG composites was assessed by coculture of rat osteoblasts in vitro. Extracts were obtained by soaking the GTG composites in deionized distilled water for 1, 2, 4, 7, 14, 28 and 42 d. The extract mixed with complete medium in a ratio of 1:1 was added into the cell culture wells containing 1 x 10(4) cells ml(-1) osteoblasts. After culturing for 2 days, the cells attached to the surface of wells were trypsinized and the number calculated by the Neubauer counting-chamber under the optical microscope. Finally, three samples in each GTG group were examined by scanning electron microscopy (SEM) to observe the morphology of the osteoblasts attached to the surfaces of GTG composites. The examinations of osteoblasts cocultured with the developed GTG composites were used to decide the ideal concentration of glutaraldehyde as a cross-linking agent. The results of extracts cocultured with osteoblasts showed that the extracts obtained from the 2, 4 and 8% glutaraldehyde cross-linked GTG composites would inhibit the growth of osteoblasts in the first 4 soaking days. During the 4-7 days soaking, the cell numbers quickly increased with the soaking time, thereafter, the cell numbers almost reached a constant value. In the analyses of substances released from the GTG composites, it was found that the gelatin and calcium were gradually released from the GTG composites, which were supposed to be nutritious for the growth of the osteoblast. The results of osteoblasts cocultured with the GTG composites showed that the concentration of glutaraldehyde used as a cross-linking agent should be lower than 8%. Compared to the GTF (composite combined with tricalcium phosphate ceramic particles and formaldehyde cross-linked gelatin), GTG composites were much suitable for a large defect bone substitute in the near future.

A study on grafting and characterization of HMDI-modified calcium hydrogenphosphate

It is known that the organic molecules can provide an effective means to manipulate the surface properties of the biodegradable ceramic. There are two ways to modify the surface of the biodegradable ceramic by organic molecules. The first one is through surface adsorption but organic molecules will easily be washed out in the physiological environment. The second approach is to graft organic molecules through covalent bond to the hydroxyl groups that are available on the surface of the ceramics. Isocyanate group has been reported as a coupling agent for hydroxyapatite and organic molecule. The studies showed that the isocyanate could react with hydroxyl groups of hydroxyapatite and form a covalent bond between isocyanate and hydroxyapatite. In the study, hexamethylene diisocyanate (HMDI) was used as coupling agent and calcium hydrogenphosphate (CaHPO4, CHP) was the candidate ceramic. CHP will react with HMDI at the temperature of 20 degrees C, 30 degrees C, 40 degrees C, 50 degrees C, 60 degrees C, and 70 degrees C for 4h. Dibutyltin dilaurate and hydroquinone were used as catalyst and inhibitor, respectively. The effect of reaction temperature on the grafted yield will be described. The linkage between CHP and HMDI will be characterized by DTA, TGA, FTIR, XRD, and 31P, 13C liquid state NMR. From the results, we successfully modified the surface of CHP with coupling agent of HMDI. The grafted yield of HMDI on CHP was increasing with the reaction temperature. The best temperature for CHP modified by HMDI is around 50 degrees C. The linkage between HMDI and the surface of CHP is a urethane linkage as CHP-O-CO-NH-(CH2)6-N=C=O. After further treatment, the terminal group of CHP treated with HMDI (MCHP) will be converted into a primary amine group as the formula of CHP-O-CO-NH-(CH2)6-NH2. If reaction temperature is 60 degrees C, long extension chain will occur with a urea linkage between the isocyanate groups as the formula of CHP-O-CO-NH-(CH2)6-(NH-CO-NH-(CH2)6)n-NH2. At reaction temperature higher than 60 degrees C, the HMDI will become prepolymerized forms in solution. The prepolymerized forms such as allophanate, biuret, uretidione and urea linkage will turn the solution into gel type mixture, which will lead to low grafted yield of HMDI on CHP. When MCHP prepared at the temperature 20 degrees C, there is no evidence of long extension but the grafted yield is the lowest only 0.9 wt% around.

The effect of morphology variety of EVAL membranes on the behavior of myoblasts in vitro

Not only the surface morphology but also the surface chemistry can be changed during the fabrication of biomaterials. Therefore, the result of a biocompatibility test of one material may alter to a great extent, dependent on the fabrication process. In this paper, the in vitro interaction of myoblasts and EVAL membranes with different surface properties was investigated. It was observed that moderate contact angle and porous structure are favourable for the cell adhesion and growth. However, cell adhesion and growth were decreased on a porous structure with particulate morphology and higher contact angle.

Biological effects and cytotoxicity of tricalcium phosphate and formaldehyde cross-linked gelatin composite

Abstract The purpose of this study was to prepare and evaluate a composite, GTF, combining tricalcium phosphate (TCP) ceramic particles and formaldehyde cross-linked gelatin, as bone substitute. The content of formaldehyde in the developed material was used to control the physical and mechanical properties of the gelatin structure, in terms of cross-linked gelatin molecules, solubility, and biodegradation of the reconstituted matrices. Formaldehyde, however, is known to be a potentially toxic substance that would cause inflammation and severe tissue response. Thus, it is necessary to know whether the substances released from the GTF composite will cause severe tissue response. In this study, the biocompatibility and cytotoxicity of the GTF composite were examined by the in vitro method of human myoblast cell culture. Extracts were obtained by soaking the GTF composite in normal saline for 1, 2, 4, 6, and 8 days. The extract mixed with complete medium in a ratio of 1:1 was added to cell culture wells each containing 1 × 10 5 myoblasts. After being cultured for 2 days, the cells were trypsinized and counted in a Neubauer countingchamber under an optical microscope. The culture dishes were washed with 0.185 M sodium cacodylate buffer (pH 7.2) and fixed with 4% glutaraldehyde solution for 30 min. They were then dehydrated through a graded ethanol series and stained with hematoxylin/eosin for optical microscopic examination. The substances released from the GTF composite were analyzed by gas chromatography (GC), capillary electrophoresis (CE), and inductive coupled plasma-atomic emission spectrometry (ICP-AES). The results showed that the GTF composite was well tolerated by the myoblast. The unchained or uncross-linked formaldehyde was completely released from the GTF composites after being soaked in the normal saline for 4 days. The extract was thought to inhibit the cell growth for the initial 4 days of soaking time. After soaking for 4–6 days, the GTF composite gradually began to release some nutritious elements which were beneficial to the myoblast growth and caused cell numbers to increase substantially. The released nutritious constituents were analyzed by the above-mentioned instruments. The results were in agreement with the above observations.

Development of biodegradable polyesterurethane membranes with different surface morphologies for the culture of osteoblasts

To evaluate the biocompatibility of biodegradable polyesterurethane membranes with different surface morphologies for their possible use as orthopedic biomaterials, rat osteoblasts were cultured on smooth, sunken, and particulate polyesterurethane membranes. A close interaction between cells and exposed particles on the particulate membranes was found. Cells on the particulate surfaces were well spread and flattened and had the greatest adhesion while cells on the smooth surfaces were more rounded, less spread, and less adhered. In addition, in order to investigate their in vivo degradation rates, the morphologic changes in retrieved membranes from 2, 4, and 8 weeks after subcutaneous implantation were observed by scanning electron microscopy and their average molecular weight changes were determined by gel permeation chromatography. These analyses showed that smooth membranes, compared with the two other surface membrane types, had the greatest rate and degree of molecular weight change. In contrast, the molecular weight of particulate membranes, which favor the osteoblast culture, had not changed significantly at 8 weeks postimplantation. Thus particulate polyesterurethane membrane surfaces may be of use as an orthopedic biomaterial, and polyesterurethane membranes certainly provide an ideal system for further study of the relative contributions to biocompatibility and degradation derived from surface morphology.

Evaluation of a novel malleable, biodegradable osteoconductive composite in a rabbit cranial defect model

Abstract The ceramic form of calcium phosphate osteoconductive material such as hydroxyapatite is brittle, non-malleable and non-degradable, and these mechanical properties limit its clinical application in calvarium reconstruction. To improve these properties, we developed a malleable, biodegradable osteoconductive composite composed of tricalcium phosphate particles bound by a gelatin which is set by glutaraldehyde mediated cross-linking. The composite was implanted into a 15 × 15 mm full-thickness, calvarial defect in 20 rabbits for up to 3 months. Twelve rabbits were left unreconstructed as controls. Specimens were retrieved at 2 weeks, 1, 2 and 3 months. Five reconstructed and 3 unreconstructed rabbits were examined for each time period. The assessment included a series of post operative gross examinations, radiographs and histologic evaluations. We are able to demonstrate that this composite is (1) biocompatible, with little tissue reaction; (2) osteoconductive, with progressive growth of new bone into the calvarial defect; (3) biodegradable, with progressive replacement of the composite by new bone, acellular matrix and bone-like material. Replacement of this composite by new bone is postulated to occur by a combination of osteoconduction and biodegradation. These results indicate that further experimental research to combine this malleable, biodegradable, osteoconductive composite with an osteoinductive agent such as bone morphogenetic protein may generate new biomaterial for full-thickness calvarial defect reconstruction.

The bonding behavior of DP-Bioglass and bone tissue

There are many reports on the surface reactions of surface-active ceramics. A Ca-P-rich layer was found on the surface of these bioactive ceramics implanted in bone tissue, a chemical bond having been established between the mineralized matrix of the bone and the apatite layer of the bioactive ceramic. It has been reported that the direct bonding of bone to DP-Bioglass was due to the deposition and subsequent mineralization of organic bone matrix at the outer layer of the implant. Thus the strength of the bonding of DP-Bioglass with bone structure is expected to be such that it will overcome the fixing problems of joint replacement and improve the long-term performance of prostheses if the bioactive glass is coated onto alloys or stainless steel. In this study, DP-Bioglass was pressed into a steel disc, 6 mm in diameter and 5 mm thick, under a hydrostatic pressure of 270 MPa, and then sintered at 810 °C for 2 hours. The DP-Bioglass discs were implanted into the condyle area of mature male rabbits for 2, 4, 8, 16 and 32 weeks. The failure load, when an implant detached from the bone or when the bone itself broke, was measured by a push-out test. Sintered hydroxyapatite bioceramic was used in a control group and the results were compared with those using DP-Bioglass. The histological evaluation and histomorphometric investigation are described in the study to demonstrate the bonding behavior between DP-Bioglass and bone tissue.

In vitro study of the effect of doxorubicin released from EVAL membrane on vesical cancer cells

A mixture of poly(ethylene-co-vinyl alcohol) (EVAL), dimethyl sulfoxide (DMSO) and doxorubicin was precipitated from the 1-octanol to form membranes with particles bonded to each other. The drug release profile from the EVAL membrane was investigated for 96 h. Results showed that the release behavior of doxorubicin was increasing with time and a two-step release behavior, a fast and a slow releasing pattern was revealed. It was also found that the number of cancer cells could be significantly decreased by released doxorubicin from the EVAL membrane. It is concluded that the EVAL copolymer could be an ideal encapsulating material for drugs to treat chronic diseases.

Biological effects and cytotoxicity of the composite combined with tricalcium phosphate and glutaraldehyde crosslinked gelatin for orthopedic application

The purpose of this study was to prepare and evaluate the feasibility and cytocompatibility of a composite (GTG) as a large defect bone substitute. The composite is combined with tricalcium phosphate ceramic particles and glutaraldehyde cross-linked gelatin. The cytotoxicity of the GTG composite were examined by extracts cocultured with rat osteoblasts in vitro. The results of extracts cocultured with osteoblasts showed that the extracts obtained from the 2%, 4%, acid 8% GTG composites after soaked for 4 days were to inhibit the growth of osteoblasts. After being soaked for 4-7 days, the cell numbers were quickly increasing with the soaking time, and then the cell number almost reached a plateau after 14-day soaking period. In the analyses of substances released from the GTG composites, the authors found that the GTG composites were gradually releasing some nutritious elements containing gelatin and calcium.