OBJECTIVETo investigate the feasibility of using marrow stromal osteoblast-cancellous bone matrix compound artificial bone (MCCAB) as tissue-engineered bone, the osteogenesis of MCCAB in the cranial defect was observed in the experiment.

METHODSThe in vitro cultivated and induced marrow stromal cells of adult New Zealand rabbits were seeded into the alginate-cancellous bone matrix to form MCCAB. The MCCAB was then implanted into the cranial defect for 4 to 8 weeks. The cancellous bone matrix (CBM) alone or the marrow stromal osteoblasts (MSOs) alone was implanted as the control. The effectiveness of bone formation was assessed by histological and roentgenographic analysis.

RESULTSThe osteogenesis of MCCAB was better than CBM or MSOs and superior to the blank group.

CONCLUSIONMCCAB can effectively repair cranial defect. It could be used clinically to restore large bone defects.

Animals ; Bone Marrow Cells ; cytology ; physiology ; Bone Matrix ; cytology ; Cells, Cultured ; Feasibility Studies ; Male ; Osteoblasts ; cytology ; physiology ; Rabbits ; Skull ; abnormalities ; Stromal Cells ; cytology ; physiology

To establish an experimental model of osteoblasts to easily cause calcification of bone matrix in vitro, we took cranium of a newborn rabbit out under an aseptic condition, removed the connective tissue of the bony suture, and cut the cranium freely into the fragments of not more than 1 mm2. The we isolated and cultured the osteoblasts using tissue explant method. We observed growth status of primary osteoblasts and subcultured osteoblasts using inverted microscope. Then we conducted enzyme staining and alizarin red staining for the third generation of osteoblasts to detect the alkaline phosphatase (ALP) expression and calcified nodules. The result showed that there were calcified nodules or calcification formed after the primary osteoblasts climbing out from the bone for 1 week, and each generation of osteoblasts had the similar calcification with the primary osteoblasts, and there was an increase in calcified nodules after the continuous culture. There was a strong expression of ALP in the plasma membrane of osteoblasts. The calcified nodules were red with alizarin red staining. It is well concluded that osteoblasts isolated with this method easily cause calcification, and can be used as a new experimental model.
Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Cell Separation ; methods ; Osteoblasts ; cytology ; Primary Cell Culture ; methods ; Rabbits ; Skull ; cytology

OBJECTIVETo study the effect of bio-derived bones, as substitutes of autogenous bone grafts and demineralized cadaver bones, on the attachment, spreading and proliferation of isolated osteoblasts.

METHODSOsteoblasts were isolated from the calvaria of a fetal rabbit through sequential collagenase digestion. In the attachment study, the osteoblasts labeled with 3H-leucine were incubated with the bio-derived bone materials in sterile microcentrifugable tubes for 15, 90 and 180 minutes, and 24 hours, respectively. The attached cells were collected and the radioactivity was measured with liquid scintillation spectrometry. In the proliferation study, the osteoblasts were cultured with the bio-derived bone materials for 24 hours and 3H-thymidine was added during the last 2 hours of the incubation. The attached cells were collected and the radioactivity was measured with liquid scintillation spectrometry. Osteoblasts were seeded on the bone graft materials for 60 or 120 minutes, 24 or 48 hours, and 3 or 7 days, then the co-culture was processed for scanning electron microscopy to observe the interaction of osteoblasts and the bio-derived bone materials.

RESULTSOsteoblasts attached to the bio-derived bone materials in a time-dependent manner. There were significantly (P<0.05) more attached cells after 180 minutes than after 15 and 90 minutes of incubations (P<0.05). Osteoblasts were proliferated in a large amount on the surface and in the materials. Osteoblasts seeded onto 100 mg bio-derived bones resulted in significantly (P<0.05) more measurable proliferation than those seeded onto 10 mg bones. Osteoblasts appeared round as they attached to the materials, then flattened and spread over with time passing.

CONCLUSIONSBio-derived bones can provide a good environment for the attachment and proliferation of osteoblasts.

Animals ; Bone Substitutes ; Cadaver ; Cell Culture Techniques ; methods ; Cell Proliferation ; Humans ; Osteoblasts ; cytology ; Osteogenesis ; Rabbits ; Skull ; cytology ; Tissue Engineering ; methods

The performance of extremely low-frequency electromagnetic fields (ELEF) can change the response of osteoblasts. In this study a kind of ELEF stimulator, which can radiate sinusoidal, rectangular, triangular and pulsed burst ELEF with frequency of 0-300 Hz and strength of 0-40 mT was developed. The neonatal rat calvarial osteoblasts were treated with the device. The effects of various ELEF stimulation on the osteoblast were evaluated by its proliferation and differentiation. The results demonstrated that rectangular and pulsed burst radiation (15Hz, 5mT) can positively change the proliferation of the osteoblast and inhibit its differentiation, but the sinusoidal one can significantly enhance its differentiation and decrease its proliferation.
Animals ; Animals, Newborn ; Cell Differentiation ; radiation effects ; Cell Proliferation ; radiation effects ; Cells, Cultured ; Electromagnetic Fields ; Osteoblasts ; cytology ; radiation effects ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology

This is an in vitro study to explore the effects of cryopreservation and resuscitation on the biological characteristics of osteoblasts at different times. Osteoblasts taken from the crania of newly born SD rats were cultured. Comparative studies were made on the cells' proliferation, the activity of alkaline phosphatase (ALP), and the number of live cells among fresh cultured osteoblasts and cells after the inception of cryopreservation and resuscitation at time-periods of one, three, six months respectively. The results showed that there were no significant differences among four groups in cell proliferation and in activity of ALP (P > 0.05). Yet, after cryopreservation and resuscitation, there were significant differences between the six-month group and the other three groups (P < 0.05). The results also showed, after cryopreservation and resuscitation, there were no significant differences between the control group and the one-month and three-month groups, respectively (P > 0.05). These findings indicated that the live cells might decrease in number after the osteoblasts were cryopreserved for too long a period, but after cryopreservation and resuscitation, the cells still retained the original biological characteristics of osteoblasts.
Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Cell Proliferation ; Cells, Cultured ; Cryopreservation ; methods ; Osteoblasts ; cytology ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; Time Factors ; Tissue Engineering ; methods

A rotating perfusion bioreactor system has recently been developed in our laboratory to produce 3D dynamic culture condition, and the critical-sized scaffolds with interconnected microchennels were fabricated. Gas exchange occurs by semipermeable membrane covered on each side of bioreactor and gas-permeable peristaltic pump tube. Rotation and perfusion of culture media through large scaffolds enhance well mixing and mass transport of oxygen and nutrients in the bioreactor. Osteoblastic cells attached to microchennels are exposed to a low fluid flow-induced shear stress level. This bioreactor system overcomes several defects exited in static culture condition, improves the culture environment, facilitates osteoblast proliferation, differntiation, significant matrix production and mineralization, and the controllability of culture process is enhanced. Large scaffolds/osteoblast constructs were cultured in the bioreactor system for 14 days. Osteoblastic cells attached to microchannels of scaffolds were observed under scanning electron microscope (SEM). The results indicated that cells grew extensively in the microchennels of large scaffolds.
Animals ; Animals, Newborn ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Cell Differentiation ; Osteoblasts ; cytology ; Rabbits ; Skull ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds

Uniaxial stretch strain and compressive pressure of 2 atm were applied to rat's osteoblasts, and then immunohistochemistrical staining was used to detect the expression of osteoblasts' c-fos gene. The experiment result indicated the osteoblasts' FOS proteins increased prominently, and the FOS proteins concentrated in nucleolus after having endured two different kinds of loadings. It is very important to prompt stress and strain in promoting osteoblast proliferation.
Animals ; Animals, Newborn ; Cell Proliferation ; Cells, Cultured ; Osteoblasts ; cytology ; metabolism ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Skull ; cytology ; Tensile Strength

This is an experimental study in the realm of physiology inquiring about the effect of pulsatile fluid flow shear stress on the proliferation, differentiation and functions of osteoblasts;the objective is to validate the important effect of fluid flow shear stress on the mechanics adaptability of bone tissue. The osteoblasts derived from Wistar rat's calvaria were exposed to fluid shear stress 5, 10, 20 and 30 mN/cm2 for 3, 6, 9, 12, 24, 36h respectively in the flow chamber. The ability of proliferation, alkaline phosphatase (ALP) activity and extracellular calcium secretion of osteoblasts were assessed. The results showed that fluid flow shear stress at 5, and 10 mN/cm2 increased the proliferation, but at 20 and 30 N/cm2, the shear stress inhibited the proliferation. The shear stress at 5, 10, 20 mN/cm2 increased the ALP activity and extracellular calcium secretion of osteoblasts, and advanced the time of the peak value of ALP activity during the experiment period, but the shear stress at 30 mN/cm2 decreased ALP activity. So osteoblasts responded rapidly to shear stress; the proliferation, differentiation and mineralization of cells were regulated in the presence of some shear stress; and such regulation exhibited a pattern of dependence on the mN/cm2 level of shear stress.
Alkaline Phosphatase ; metabolism ; Animals ; Cell Proliferation ; Cells, Cultured ; Mechanotransduction, Cellular ; physiology ; Osteoblasts ; cytology ; enzymology ; Pulsatile Flow ; Rats ; Rats, Wistar ; Shear Strength ; Skull ; cytology ; Stress, Mechanical

OBJECTIVETo observe the effects of two main isoflavones, daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro.

METHODSOsteoblasts obtained from newborn Sprague-dawley rat calvaria were cultured for several generations. The second generation cells were cultured in Minimum Essential Medium supplemented with ascorbic acid and Na-beta-glycerophosphate for several days, in the presence of daidzein and genistein, with or without the estrogen receptor antagonist ICI 182780. Number of nodules was counted at the end of the incubation period (day 20) by staining with Alizarin Red S calcium stain. The release of osteocalcin, as a marker of osteoblast activity, was also determined on day 7 and day 12 during the incubation period.

RESULTSCompared with the control, the numbers of nodules were both increased by incubation with daidzein and genistein. 17 beta-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation that daidzein and genistein. The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein, as well as 17 beta-estradiol on day 7 and day 12 (day 12 were higher). The estrogen receptor antagonist ICI 182780 completely blocked the genistein- and 17 beta-estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts. However, the effects induced by daidzein could not be inhibited by ICI 182780.

CONCLUSIONThese findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts. The effects, like those induced by 17 beta-estradiol, are mediated by the estrogen receptor dependent pathway. Daidzein also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts, but it is not, at least not merely, mediated by the estrogen receptor dependent pathway.

Animals ; Cells, Cultured ; Genistein ; pharmacology ; Isoflavones ; pharmacology ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteocalcin ; biosynthesis ; Osteogenesis ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; drug effects ; metabolism

3D Scaffolds with controlled porous structure were designed and fabricated by utilizing CAD and rapid prototyping techniques. A flow perfusion bioreactor, which allowed exposure of the culture cells to low levels of mechanical stimulation by fluid flow-induced shear stress, was developed in our lab. The scaffolds were pre-designed and the negative images of the designs were used to build the molds on a stereolithography (SL) apparatus with epoxy resins. Calcium phosphate cement paste was cast into the molds. And after pyrolysis, the 3D scaffolds with controlled internal pore architectures were obtained. Rabbit osteoblasts were seeded in 3D porous scaffolds, cultured in the flow perfusion bioreactor with media flow rate set at 2 mL/min and 6-well plates. At 3, 7, and 14 days, scanning microscopic evaluation showed excellent growth on the surface of scaffolds and poor viability of cells within microchannels in static cultures. In flow perfusion bioreactor, there was greater cellularity throughout the scaffolds and abundant deposition of extracellular matrix. Cells were also seen throughout the internal microchannels of scaffolds. These results represent that better mass transport of oxygen and nutrient occurred in the flow perfusion bioreactor and cells distribution in 3D porous scaffolds was improved.
Animals ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Cell Division ; Cells, Cultured ; Osteoblasts ; cytology ; Porosity ; Rabbits ; Skull ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds