ABSTRACT
The preparation of collagen sponges was studied in order to develop tissue engineering scaffolds. Collagen solutions with varying concentrations were obtained by condensing the initial collagen with polyethylene glycol (PEG) at 4 degrees C for different periods of time, and then were freeze-dried to make collagen scaffolds. The porous characteristics of the prepared scaffolds were characterized by use of different methods, including laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM) and tensile tests. All collagen sponges were shown to have similar interconnected porous structures but were found to have different pore size, porosity, water capacity and the mechanical property, depending on the concentration of collagen solutions. These findings indicate that the way of controlling the concentration of collagen solutions with PEG permits the freeze-drying fabrication of collagen sponges with varying porous features suitable for different tissue engineering purposes.
Subject(s)
Collagen , Chemistry , Freeze Drying , Polyethylene Glycols , Chemistry , Porosity , Tissue Engineering , Tissue ScaffoldsABSTRACT
BACKGROUND: Up to date, studies concerning capspase 3 inhibitor mainly focus on peptide/non-peptide compounds synthesis and detection. Few reports addressing inhibits chondrocytes apoptosis using silenced caspase 3 gene. OBJECTIVE: To inhibit apoptosis of chondrocytes by blocking the apoptotic cascade reaction, gene silencing of caspase 3, and transduction of caspase 3 siRNA into chondrocytes with lentivirus vector.DESIGN, TIME AND SETTING: A single sample observation was performed at the Institute of Traumatic Surgery, Guangzhou Red Cross Hospital, Medical College of Jinan University from June 2008 to June 2009.MATERIALS: Chondrocytes were harvested from SD rats, and caspase 3 shRNA plasimid was constructed by our laboratory.METHODS: Rattus caspase 3 siRNA was synthesized and cloned into pSIH1-H1-copGFP plasmid. pSIH1-H1-copGFP-caspase 3 siRNA lentivirus was generated in 293TN cells by pPACKH1~(TM) Lentivector Packaging Kit and transducted into chondrocytes of rats.MAIN OUTCOME MEASURES: After the lentivirus was transducted into chondrocytes, the caspase 3 mRNA was tested by RT-PCR and the caspase 3 protein was tested by Western blot. Both the transducted cells and untransducted cells were induced apoptosis by tumor necrosis factor α (TNF-α). Cell apoptosis was assessed by flow cytometry, Annevin V/PI.RESULTS: The transduction rate of caspase 3 siRNA was about 90% by lentivirus vector. The expression of caspase 3 mRNA and caspase 3 protein in transducted chondrocytes was lower than the normal chondrocytes (P < 0.01). When the cells induced apoptosis by TNF-α, the apoptosis rate of the negative siRNA- chondrocytes was 7 times higher than that of caspase 3 siRNA-chondrocytes.CONCLUSION: The caspase 3 siRNA could inhibit caspase 3 expression and decrease drug-induced apoptosis of the chondrocytes.
ABSTRACT
This study sought to explore a new compound polyvinyl alcohol-collagen as a wound dressing. To make the polymer, Polyvinyl alcohol (PVA) and collagen type I were put together in the ratio of 3:1, at the same time, polyethlene glycol as porogen was added, and the material was dried by air to be a membrane in shape. Then the ultimate tensile load, the hole diameter, porosity, and water absorption were measured. The cell biocompatibility was tested as well. The results showed the PVA-collagen blend had the average hole 100-150 microm in diameter, and the porosity about 90%. The ultimate tensile load reached 8.10 +/- 0.28 MPa, and water absorption was up to 185.42% +/- 6.93%. 3T3 cells grew well on the PVA-collagen member. Therefore, the PVA-collagen memberane is characterized not only by its ideal biomechanical ability and biocompability, but also by its ideal hole diameter, porosity and water absorption. It may have the potential for use as a wound dressing in vivo.
Subject(s)
Humans , Biocompatible Materials , Chemistry , Collagen , Chemistry , Occlusive Dressings , Polyvinyl Alcohol , ChemistryABSTRACT
BACKGROUND: Polyvinyl alcohol (PVA) displays limitation to cell adsorbability. Can collagen improve the adsorbability of PVA to cells?OBJECTIVE: To develop a novel type composite of PVA and collagen, and explore the feasibility to serve as soft tissue substitute.DESIGN: Single sample observation.SETTING: Guangzhou Red Cross Hospital, Jinan University Medical College, Guangzhou Institute of Traumatic Surgery.MATERIALS: Fifteen New Zealand rabbits of 2.0-3.0 kg, either male or female, were provided by Medical Experimental Animal Center of Guangdong Province. The experiment was carried out in the Laboratory of Guangzhou Institute of Traumatic Surgery, and the experimental procedure was accorded with the animal ethical standards. Bovine typeI collagen was purchased from Guangzhou Trauer Biotechnology Co., Ltd. and PVA-124 from Guangzhou Chemical Reagent and Instrumentation Co., Ltd.METHODS: The experiment was performed in Guangzhou Red Cross Hospital, Jinan University Medical College between July 2003 and December 2006. ①Preparation of PVA-collagen material: 5 g/L bovine type I collagen was mixed with 5% PVA-124 at a ratio of 1 : 1. The mixture was freeze-dried at vacuum until becoming gelatinous. The internal structure was observed under the use of scanning electron microscope. ②Cytotoxicity test: PVA-collagen composite was cut into pieces of 10 mm×5 mm× 1 mm, put into 48-well culture plate after sterilized by Y ray, cultured with 1×104 3T3 cells in each well. Cell growth was observed under scanning electron microscope and laser scanning confocal microscope. ③Embedding test in vivo: Two longitudinal incisions were cut at the two sides of spine. The subcutaneous tissue was separated bluntly to form subcutaneous lacuna. Four pieces of PVA-collagen material were implanted in the lacuna and fixed. Nine specimens and the surrounding tissues were harvested from three rabbits each at one, four, eight and sixteen weeks postoperatively for pathological observation.MAIN OUTCOME MEASURES: The internal structure of gel film under scanning electron microscope, cytotoxicity test and embedding test in vivo results.RESULTS: ①Internal structure of PVA-collagen material:PVA-collagen material showed white gel shape after freeze-drying at vacuum. Penetrating three-dimensional pores were observed in the surface and inner section under scanning electron microscope. ②Cytotoxicity test results showed that 3T3 cells grew normally on the PVA-collagen material. ? Embedding test in vivo results suggested that one week after PVA-collagen implantation, foreign body reaction occurred, and the interface between material and tissue was clear. Four weeks later, only rare lymphocyte infiltration was observed, and a great amount of fibroblast hyperplasia formed collagen fibrils and false simple cuboidal epithelium coating material. In 8 weeks, no lymphocyte infiltration, neutrophilic granulocyte infiltration or foreign body giant cell were found; dense capsule wall and capsule coating material generated from a great amount of fibroblasts were observed. In 16 weeks, extending collagen fibrils were found arranged regularly with shrank nucleus, showing long ovoid or long fusiform in shape; no new formation small vessels, lymphocyte, neutrophilic granulocyte infiltration or foreign body giant cell infiltration were observed. The capsule wall was stable and thinned. CONCLUSION: PVA-collagen composite has good cell compatibility and tissue compatibility but no toxic or adverse effect. It can serve as in vivo implant.
ABSTRACT
BACKGROUND: Simple polyvinyl alcohol (PVA) has limited ability to cell adhesion. There are not generally accepted studies on improved effects of collagen protein modified polyvinyl alcohol on cell adhesion and proliferation.OBJECTIVE: To investigate the feasibility of PVA/type Ⅰ college (COL-Ⅰ) as anterior cruciate ligament (ACL) scaffolds in tissue engineering.DESIGN, TIME AND SETTING: The controlled observation experiment was performed at the Fourth Affiliated Hospital, Medical College. Ji'nan University, Guangzhou Red Cross Hospital, Guangzhou Institute of Trauma Surgery from August 2006 to October 2007.MATERIALS: COL-Ⅰ gel was produced by Guangzhou Institute of Trauma Surgery.METHODS: PVA filature was used to weave fascicular scaffolds. NIH-3T3 cell line and human ACL cells were in vitro incubated, amplified, and then implanted on the PVA/COL scaffolds.MAIN OUTCOME MEASURES: The growth of NIH-3T3 cell line and human ACL cells on the PVA/COL scaffolds and the secretion of extracellular matrix were observed using scanning electron microscope. Cell compatibility of PVA/COL scaffolds was assessed. Mechanics characteristic of PVA/COL scaffolds was measured by using the electric. tensile force apparatus. Mechanical property of PVA/COL scaffolds was analyzed using the SPSS 11.5 software package.RESULTS: NIH-3T3 cell line and human ACL cells on the PVA/COL scaffolds adhered, proliferated, and secreted extracellular matrix. NIH-3T3 cell line highly grew compared with human ACL cells on the PVA/COL scaffolds. The adhered number of NIH-3T3 cell line and human ACL cells was significantly increased on the PVA/COL scaffolds. NIH-3T3 cell line and human ACL cells presented well morphology on the PVA/COL scaffolds. COL-Ⅰ could promote the secretion of extracellular matrix from NIH-3T3 cells, but its effects on human ACL cells were not significant. Tensile force test showed that load-extension curve of the materials was identical to ACL of human and rabbits, and the scaffolds possessed strong flexibility. The maximal load, ultimate stress and elastic modulus were respectively 52.61 N, 14.96 MPa and 202.08 MPa.CONCLUSION: COL-Ⅰ accelerates the adhesion and proliferation of NIH-3T3 cell line and human ACL cells on the surface and in the pore of the PVA/COL scaffolds, promotes the secretion of extracellular matrix from NIH-3T3, and PVA filature material has mechanical property and good cell compatibility.
ABSTRACT
[Objective]To investigate the feasibility of the fabrication of tissue-engineered anterior cruciate ligament(ACL)in vitro by studying biocompatibility and mechanical property of the braided polyvinyl alcohol(PVA)materials.[Method]Firstly,human ACL cells and NIH3T3 cells were isolated,expanded in vitro and seeded onto the surface of the braided PVA scaffold materials,the adhesion,proliferation and three-dimensional growth of cells on the scaffold were observed by SEM.Secondly,the biomechanical properties of the braided PVA scaffold materials were measured with electro-biomechanical machine.[Result]The braided PVA scaffold materials had no cytotoxicity,ACL and NIH3T3 cells adhered,grew and proliferated well both on the surface and in the holes of the braided PVA scaffold materials.The maximum load,the maximum strain and ultimate tensile stress of the braided PVA scaffold materials respectively were 169.78?9.18N,11.67?1.38% and 52.21?2.88MPa.[Conclusion]The braided PVA scaffold materials possess good biomechanical properties and biocompatibility,it may become an ideal biomaterial for fabricating tissue-engineering ACL if the biomechanical properties can be improved.
ABSTRACT
BACKGROUND: The repair of articular cartilage defect is always a problem that is dedicatedly solved by doctors of orthopaedics. Autologous perichondrium, periosteum or allografting of cartilage have been used previously; however, the source of the donor is limited, the fixation is difficult as well as the occurrence of endochondrial ossification and delamination between the inferior cartilage and reparative cartilage, etc. Type Ⅱ collagen, the main component of cartilage matrix, has certain effects in the repair of articular cartilage defect.OBJECTIVE: To investigate the effects of type Ⅱ collagen sponge filling on the repair of articular cartilage defect.DESIGN: A randomized controlled trial.SETTING and MATERIALS: The study was conducted in the Guangzhou Institute of Traumatic Surgery. Materials were 24 adult male purebred New Zealand Rabbits(48 knees), ordinary grade, with a body mass of (2.29 ±0. 25) kg. Animals were fed with standard feeding in separate cage.INTERVENTIONS: A full-thickness defect in articular cartilage was made on the femoral trochlear surface by a drill of 5 mm in diameter and 3 mm in depth. Rabbits were allocated into filling group(type Ⅱ collagen sponge was grafted into left keen joint defect) and control group(right knee joint defect site was set as control) according to random number table.MAIN OUTCOME MEASURES: Gross morphological and histological observation of the defect repair in each dual week within 12 weeks after operation.RESULTS: During 10 - 12 weeks, in cuntrol group: The defect area was repaired by white and soft tissue that had no resistance to press. The repaired tissue was still lower than the surrounding articular surface with clear boundary. By histological observation, it was found that the defect was repaired by the mechanism similar to inflammatory reaction and the defect is ultimately filled by the hyperplasia of hyaline degenerative fibrous tissues. In filling group: the defect was repaired by semi-transparent, smooth, textured tissues with polish that had resistance to press as well as elasticity. The repaired tissue was almost similar to the shape of the surrounding cartilage,difficult to be distinguished. After histological observation, it was found that there was no inflammatory reaction, but active hyperplasia of inner bonetissue and cartilage tissues; a lot of osteoid tissues and trabeculation were found. Newlborn cartilage was fused with surrounding cartilage tissue and connected with surrounding tissues. Type Ⅱ collagen had significantly promoting effects in the repair of articular cartilage defect, and the repaired cartilage was close to normal cartilage. CONCLUSION: The self-made high purity type Ⅱ collagen sponge has favorable promoting effects in the repair of articular cartilage defect with good histocompatibility but without obvious toxic side effect.
ABSTRACT
We have prepared a wound dressing made from chitosan and collagen. Its clinical curative effect was detected. Chitosan solution was put into purified collagen solution. Then, the solution became sponge by means of freeze drying, and it was subjected to a series of toxicology tests, including acute toxicity, stimulation test, allergic and hemolysis tests, as well as the clinical test of openning trauma in orthopedics. All of the results of toxicology tests were negative. The chitosan-collagen sponge could not only accelerate the speed of curing but also restrain the extravasate. Therefore, the chitosan-collagen sponge has good biocompatibility and clinical curative effect. It is a prospective security-biomaterial for medical use.