Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.
MeSH terms: Animals; Biocompatible Materials/toxicity*; Cell Survival/drug effects; DNA Damage*; Hydroxyapatites/toxicity*; L Cells (Cell Line); Mutagenicity Tests*; Bone Substitutes/toxicity*; Prostheses and Implants*; Mice
The aim of this study was to evaluate the in vitro cytotoxicity of biomaterials; Hydroxyapatite (HA), Natural coral (NC) and Polyhydroxybutarate (PHB). Three different materials used in this study; HA (Ca10(PO4)6(OH)2), NC (CaCO3) and PHB (Polymer) were locally produced by the groups of researcher from Universiti Sains Malaysia. The materials were separately extracted in the complete culture medium (100mg/ml) for 72h and introduced to the osteoblast cells CRL-1543. The viability of osteoblast CRL-1543 cultivated with these extraction materials after 72h incubation period was compared to negative control with neutral red assay by using spectrophotometer at 540nm. The results showed the non-cytotoxicity of the materials. After 72h of incubation period, HA showed 123% viable cells, NC was 99.43% and PHB was 176.75%. In this study, cytotoxicity test dealt mainly with the substances that leached out from the biomaterial. The results obtained showed that the materials were not toxic and also promoted cells growth in the sense of biofunctionality.
Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.
MeSH terms: Animals; Biocompatible Materials/toxicity*; L Cells (Cell Line); Cell Death/drug effects*; Apoptosis/drug effects; Durapatite/toxicity*; Bone Substitutes/toxicity*; Prostheses and Implants*; Mice
The study was carried out to evaluate macroscopically the ability of coral to repair a large size bone defect. A total 12 adult, male sheep were used in the study. The large bone defect (2.5cm x 0.5cm x 0.5cm) was created surgically on the left proximal femur and replaced by a block of coral (Porites sp.). Radiographs were obtained immediately after surgery and at 2, 4, 8 and 12 weeks post-implantation. Ultrasonographic examinations were carried out every 2 weeks after implantation up to 12 weeks using ultrasound machine (TOSHIBA Capasee II) connected with 7MHz frequency transducer. The sheep were euthanased at 2, 4, 8, and 12 weeks post-implantation and the bone examined grossly. Both ultrasonographs and radiographs taken at 8 and 12 weeks showed that the implants had been resorbed and left the space that much reduced in size. There was no sign of implant rejection observed in all animals. The results showed that processed coral has potential to become bone substitute for reconstructive bone surgery.
MeSH terms: Animals; Biodegradation, Environmental; Bone and Bones/pathology*; Calcium Carbonate/analysis*; Femur/pathology; Male; Radiography*; Sheep; Ultrasonography*; Osseointegration/physiology*; Bone Substitutes/analysis*; Absorbable Implants*; Anthozoa*
Failure analysis was performed to investigate the failure of the femur fixation plate which was previously fixed on the femur of a girl. Radiography, metallography, fractography and mechanical testing were conducted in this study. The results show that the failure was due to the formation of notches on the femur plate. These notches act as stress raisers from where the cracks start to propagate. Finally fracture occurred on the femur plate and subsequently, the plate failed.
Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
MeSH terms: Adolescent; Adult; Age Factors; Aged; Burns/physiopathology; Burns/therapy*; Cell Division/physiology; Cell Survival/physiology; Child; Chronic Disease; Female; Humans; Male; Middle Aged; Statistics as Topic; Wound Healing/physiology; Wounds and Injuries/physiopathology; Wounds and Injuries/therapy*; Keratinocytes/cytology*; Skin Transplantation*; Cell Aging/physiology*; Tissue Engineering/methods*
Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) evaluation were carried out in the in vivo skin construct using fibrin as biomaterial. To investigate its progressive remodeling, nude mice were grafted and the Extracellular Matrix (ECM) components were studied at four and eight weeks post-grafting. It was discovered that by 4 weeks of remodeling the skin construct acquired its native structure.
Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.
MeSH terms: Cell Adhesion/physiology*; Collagen*; Energy Metabolism/physiology; Fibroblasts/cytology; Humans; Microscopy, Electron, Scanning; Organ Culture Techniques/methods*; Polymers*; Tissue Engineering/methods*; Chitosan*
Cartilage is regularly needed for reconstructive surgery. Basic research in tissue engineering is necessary to develop its full potential. We presented here the expression profile of type II collagen gene and type I collagen gene in human auricular monolayer culture expansion. Cultured chondrocytes documented a reduction in the expression level of collagen type II gene whilst collagen type I gene was gradually expressed through all the passages. This study demonstrated that human auricular chondrocytes lose its phenotypic expression during monolayer culture expansion. Further studies are required to enhance cartilage specific gene expression, collagen type II throughout the in vitro culture.
This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.
To date there is no optimal approach to reconstruct an external ear. However, advances in tissue engineering technologies have indicated that in vitro autologous elastic cartilage might be of great importance in the future treatment of these patients. The aim of this study was to observe monolayer expansion of auricular cartilage and to evaluate engineered cartilage using standard histochemical study.
We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.
Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be selectively isolated and induced to express bone phenotype in vitro. The osteoblastic progenitor can be estimated by counting the number of cells attach using the haemacytometer. This study was undertaken to test the hypothesis that human aging is associated with a significant change on the number of osteoblastic progenitors in the bone marrow. Bone marrow aspirates were harvested from 38 patients, 14 men (age 11-70) and 24 women (age 10-70) and cultured in F12: DMEM (1:1). In total 15 bone marrow samples have been isolated from patients above 40 years old (men/women) of age. Fourteen (93.3%) of this samples failed to proliferate. Only one (6.7%) bone marrow sample from a male patient, aged 59 years old was successfully cultured. Seventy percent (16/23) of the samples from patient below than 40 years old were successfully cultured. However, our observation on the survival rate for cells of different gender from patient below 40 years old does not indicate any significant difference. From this study, we conclude that the growth of bone marrow stromal cells possibly for bone engineering is better from bone marrow aspirates of younger patient.
Tricalcium phosphate/hydroxyapatite (TCP/HA), hydroxyapatite (HA), chitosan and calcium sulphate (CaSO4) were studied and evaluated for possible bone tissue engineered construct acting as good support for osteogenic cells to proliferate, differentiate, and eventually spread and integrate into the scaffold. Surface morphology visualized by SEM showed that scaffold materials with additional fibrin had more cell densities attached than those without, depicting that the presence of fibrin and collagen fibers were truly a favourite choice of cells to attach. In comparison of various biomaterials used incorporated with fibrin, TCP/HA had the most cluster of cells attached.
In the present study, natural coral of porites species was used as scaffold combined with in vitro expanded bone marrow stem cell derived osteoblasts (BMSC-DO), to develop a tissue-engineered bone graft in a rat model. Coral was molded into the shape of rat mandible seeded with 5x10(6) /ml BMSC-DO subsequently implanted subcutaneously in the back of 5 week Sprague dawely rats for 3 months. Coral alone was implanted as a control. The implants were harvest and processed for gross inspection and histological observations. The results showed that newly bone grafts were successfully formed coral seeded with cells group showed smooth highly vascularized like bone tissue. Histological sections revealed mature bone formation and lots of blood vessel, the bone formation occurred in the manner resemble intramembraneous bone formation. This study demonstrates that coral can be use as a suitable scaffold material for delivering bone marrow mesenchymal stem cells in tissue engineering.
MeSH terms: Animals; Biodegradation, Environmental; Bone Marrow Cells/cytology*; Calcium Carbonate*; Microscopy, Electron, Scanning; Organ Culture Techniques/methods*; Osteoblasts/cytology*; Bone Transplantation*; Tissue Engineering/methods*; Anthozoa*; Rats; Mesenchymal Stromal Cells/cytology*
In this study the surface properties of two particulate coral and polyhydroxybutrate (PHB) were studied in order to characterize them prior to use in composite production. Coral powder and PHB particle were evaluated using scanning electron microscopy and confocal laser scanning microscopy, to measure surface porosity and pores size. The results showed that coral powder has multiple pleomorphic micropores cross each others give appearance of micro-interconnectivity. Some pore reached to 18 microm with an average porosity of 70%. PHB revealed multiple different size pores extended to the depth, with an average some times reach 25 microm and porosity 45%. These findings demonstrate that both coral and PHB have excellent pores size and porosity that facilitate bone in growth, vascular invasion and bone development. We believe that incorporation of coral powder into PHB will make an excellent composite scaffold for tissue engineering.
Selections of collagen available commercially were tested for their biocompatibility as scaffold to promote cell growth in vitro via simple collagen fast test and cultivation of mammalian cells on the selected type of collagen. It was found that collagen type C9791 promotes the highest degree of aggregation as well as cells growth. This preliminary study also indicated potential use of collagen as scaffold in engineered tissue.
A prospective study of 25 patients with supracondylar and intercondylar fracture of the femur was carried out from January 1999 to June 2003 at SSG Hospital, Baroda, India. Twenty-eight percent fractures were open and 72% were closed. Twelve pecent of the fractures had intraarticular extension. AO classification was used to classify the fractures. Eighty-four percent of the fractures were due to high velocity trauma. All the cases were operated by percutaneous supracondylar nailing. No postoperative complication related to fracture treatment was seen. Average follow up was 20.1 months (range 6-40 months). Average age of the patients was 31.5 years. Average time of union was 3.1 months (range 2-4 months). Average knee range of motion was 117 degrees. Even in open injuries, 85.7% had more than 110 infinity range of motion. All patients could return to their pre-injury lifestyle. Rating scale developed by Hospital for Special Surgery was used to quantify the results. Eighty-four percent showed excellent, 8% showed good and 8% showed fair results. Percutaneous supracondylar nailing is thus, an excellent method of treating fractures of distal femur.
Neem, Azadirachta indica, is a plant from the family Meliaceae, known as "Pokok Semambu" in Malay community. It has been extensively used in India as traditional Ayurvedic and folklore minedicine for the treatment of various diseases. This study aimed to determine the distribution of selenium in the liver of rats during hepatocarcinogenesis when neem aqueous extract and dietary selenium was supplemented.