The Growth of Osteoblasts according to the Pretreatment of HA Scaffold Surface

PURPOSE: Recently, bioceramics have become popular as a substitute graft material for reconstruction of bony defect after trauma or tumor surgery. Among the bioceramic materials, hydroxyapatite(HA) is favored due to its biocompatibility. HA scaffold is composed of the interconnected reticular framework, macropores and micropores. Macropores play an important role in cell migration, nutrients supply and vascular ingrowth. On the other hand, a number of micropores less than 10micrometer form an irregular surface on HA scaffolds, which prevents the osteoblast from adhering and proliferating on the surface of HA scaffold. METHODS: In this study, three different groups were designed for comparison. In the first group(group A), conventional method was used, in which HA pellet was applied without surface pretreatment. The second group (group B) was given a HA pellet that has been coated with crystalline HA solution prior to application. In the third group(group C), the same method was used as the second group, where the pretreated HA pellet was heated(1250degrees C, 1 hour) before application. Osteoblast-like cells(2x10(4)/mL) were scattered onto every pellet, then they were incubated in 5% CO2 incubator at 37degrees C for twelve days. During the first three days, osteoblast cells were counted using the hemocytometer daily. ALP activity was measured on the 3, 6, 9 and 12 culture days using the spectrophotometer. RESULTS: Under SEM, group A showed a surface with numerous micropores, and group B revealed more rough crystal surface. Group C revealed a fused crystal appearance and flattened smooth surface. In proliferation and ALP activity of osteoblast cells, group C showed better results compared to group B. Group A which lacks pretreatment of the surface showed less osteoblast proliferation and ALP activity than group C, but showed better results than group B. CONCLUSION: We found that crystallized HA with heat treatment method enhances the osteoblasts proliferation and differentiation on the surface of HA pellets.

The Effect of Collagen Coating in Crystallized Hydroxyapatite Scaffold

PURPOSE: Hydroxyapatite (HA) has been widely used due to its chemical similarity to bone and good biocompatibility. HA is composed of macropores and micropores. Too much irregularities of the micropores are ineffective against the adhesion and proliferation of osteoblast. Many efforts have been tried to overcome these drawbacks. HA crystal coating on the irregular surface of HA scaffold, crystallized HA, is one of the method to improve cell adhesion. Meanwhile, the collagen has been incorporated with HA to create composite scaffold that chemically resembles the natural extracellular matrix components of bone. The authors proposed to examine the effect of collagen-coated crystallized HA on the adhesion and proliferation of osteoblast. METHODS: HA powder containing 10 microM pore size was manufactured as 1 cm pellet size. In order to make crystallized HA, 0.1 M EDTA solution was used to dissolve HA powder and heated 100degreesC for 48 hours. Next, the crystallized HA pellets were coated with collagen (0.1, 0.5, and 1%). The osteoblasts were seeded into HA pellets and incubated for the various times (1, 5, and 9 days). After the indicating days, methylthiazol tetrazolium (MTT) assay was performed for cell proliferation and alkaline phosphatase (ALP) activity was measured for bone formation. RESULTS: In SEM study, the surface of crystallized HA pellet was more regular than HA pellet. MTT assay showed that the proliferation of osteoblasts increased in a collagen dose-dependent and time-dependent manner and had a maximum effect at 1% collagen concentration. ALP activity also increased in a collagen dose-dependent manner and had a highest effect at 1% collagen concentration. CONCLUSION: These data showed that crystallization and collagen coating of HA was effective for osteoblast proliferation and ALP activity. Therefore, our results suggest that crystallized-HA scaffold with collagen coating is may be a good strategy for tissue engineering application for bone formation

Radioopacity of Absorbable Plate containing Hydroxyapatite and Gold(Au); A Preliminary Report

PURPOSE: Absorbable bone fixation materials for operation of facial bone fracture are composed of poly- lactic acid(PLA) & poly-glycolic acid(PGA). These materials are absorbed after facial bone healing period. Therefore, these materials are harmless in human body. But because of it's radioopacity, the number and the location of the materials are not checked in follow-up X-ray examination. We studied absorbable bone fixation materials checked radiological examination. So, we made the absorbable plate composed of PLA, Hydroxyapatite (HA) and Gold(Au). METHODS: Plate 1 was consisted of pure PLA. Plate 2 was consisted of PLA(50%) and HA(50%). Plate 3-7 were consisted of PLA(50%), and variable composition of HA & Au. The ratio of Au was as following. From the plate 3 to plate 7, the Au ratio was 1%, 5%, 10%, 17%, and 25%, respectively. Total 3 examinations were used -naked eye examination, simple X-ray examination, and Haunsfield unit of plate in CT examination. RESULTS: Naked eye examination found out that the color of plate 1 was most white. As the Au ratio increases, the color of plate was getting close to khaki color. the radioopacity of plate 2 was similar cortical bone of face in simple X-ray. The Haunsfield unit of cortical bone of face was 1000 HU. Haunsfield unit of titanium plate was 2900 HU. Haunsfield unit of plate 1 through plate 7 were -242, 1489, 1776, 3052, 3092, 3095, and 3095, respectively. CONCLUSION: Radioopacity of plate 2 was similar to cortical bone of face. In CT examination, Hanusfield unit of plate 2 was similar to Hanusfield unit of cortical bone of face. Hanusfield unit of plate 4-7 were similar to Hanusfield unit of titanium plate. So to trace bone fixation materials after facial bone surgery, the best ratio of Au is about 1-5%. If this study is applied to facial bone surgery, radiologic follow up would be easy after facial bone surgery.

The Effects of the Mixture of Fetal Bovine Serum and Poly-glycolic acid in Rabbit Calvarial Model

PURPOSE: This study was undertaken to investigate the osteogenic induction potential of PGA & FBS mixture on a calvarial defect in the rabbit. METHODS: Twenty New zealand white rabbit, weighing from 3.5-4kg were allocated into each of the three groups. Four 8mm sized bone defects were made on the parietal bone by drilling. In group I, the bony defects were implanted with 50 micrometer thickness film containing mixture of PGA and FBS. In group II, with PGA only film, & in group III, the bony defects were left with no implants. Results were evaluated by using morphologic change, radiographic study, biochemical study and histologic examination at 1 week (group I n=7, group II n=7, group III n=14), 2 weeks (group I n=6, group II n=6, group III n=12) and 3 weeks (group I n=7, group II n=7, group III n=14) following implantation. RESULTS: In the morphologic & radiographic study, the formation and corticalization of callus were observed earlier in group I than in groups II and III (p<0.05). In histological examination, group I showed more abundant and faster new bone formation than in group II and III. In biochemical analysis, group I displayed more activity than in group II and III. Group I also showed more abundant osteopontin, osteocalcin than groups II and III. CONCLUSION: In conclusion, the results demonstrate that the mixture of PGA and FBS has an effect on osteoblastic formation in the rabbit model. It is considered that further evaluation of long term results on resorption, immunologic tissue reaction and response of applied mixture in the human model will be needed.

The Effects of the Mixture of Fetal Bovine Serum and Poly-glycolic acid in Rabbit Calvarial Model

PURPOSE: This study was undertaken to investigate the osteogenic induction potential of PGA & FBS mixture on a calvarial defect in the rabbit. METHODS: Twenty New zealand white rabbit, weighing from 3.5-4kg were allocated into each of the three groups. Four 8mm sized bone defects were made on the parietal bone by drilling. In group I, the bony defects were implanted with 50 micrometer thickness film containing mixture of PGA and FBS. In group II, with PGA only film, & in group III, the bony defects were left with no implants. Results were evaluated by using morphologic change, radiographic study, biochemical study and histologic examination at 1 week (group I n=7, group II n=7, group III n=14), 2 weeks (group I n=6, group II n=6, group III n=12) and 3 weeks (group I n=7, group II n=7, group III n=14) following implantation. RESULTS: In the morphologic & radiographic study, the formation and corticalization of callus were observed earlier in group I than in groups II and III (p<0.05). In histological examination, group I showed more abundant and faster new bone formation than in group II and III. In biochemical analysis, group I displayed more activity than in group II and III. Group I also showed more abundant osteopontin, osteocalcin than groups II and III. CONCLUSION: In conclusion, the results demonstrate that the mixture of PGA and FBS has an effect on osteoblastic formation in the rabbit model. It is considered that further evaluation of long term results on resorption, immunologic tissue reaction and response of applied mixture in the human model will be needed.

The Growth of Human Osteoblasts in Culture Dishes Made with Poly-glycolic Acid Containing Fetal Bovine Serum

PURPOSE: An ideal bony construct can be divided into two broad categories: (1) the design and fabrication of biodegradable, biomimetic scaffolds that provide correct signals to induce osteogenesis: (2) the identification of an ideal source of osteoprogenitor cells to seed onto the scaffold. We selected poly-glycolic acid as a synthetic scaffold among various scaffolds because of these properties. Meanwhile, culture medium is supplemented with fetal bovine serum(FBS): such serum contains essential elements such as proteins, hormones, growth factors and trace minerals. The composition of FBS can be ideal for various cell growth in vitro. We supposed that we could enhance bone growth at a fractured site if FBS was mixed with synthetic scaffold-PGA. METHODS: We cultured human osteoblasts in five different prepared culture dishes made with FBS and PGA mixture. The mixtures contained different ratio of FBS, that is, 0, 1.5, 3, 7, and 10%. We cultured human osteoblasts for seven days and examined the growth and attachment of the cells at the 1st, 3rd, 5th, 7th days, respectively. RESULTS: In the mixture of 0% FBS and PGA, the growth of the cells lasted for one day. In 1.5 and 3% FBS and PGA, the growth of the cells was examined at the 3rd day, then minimally declined at the 5th and 7th days. In 7% FBS and PGA, the growth of the cells lasted for 5 days, then declined at the 7th day. In 10% FBS and PGA, the growth of the cells lasted for 5 days, then declined at the 7th day. Staining status of the osteoblasts with alkaline phosphatase showed pale pink color in 0% FBS and PGA groups, but bright pink color in 1.5, 3, 7, 10% FBS and PGA groups, especially in 3%, 7%. CONCLUSION: In consequence, the growth of human osteoblast was higher in the mixture of FBS and PGA groups than in pure PGA ones. It is assumed that the mixture of FBS and PGA affects the proliferation of human osteoblasts.