A Three-Dimensional Printed Polycaprolactone-Biphasic-Calcium-Phosphate Scaffold Combined with Adipose-Derived Stem Cells Cultured in Xenogeneic Serum-Free Media for the Treatment of Bone Defects.

The efficacy of a three-dimensional printed polycaprolactone-biphasic-calcium-phosphate scaffold (PCL-BCP TDP scaffold) seeded with adipose-derived stem cells (ADSCs), which were cultured in xenogeneic serum-free media (XSFM) to enhance bone formation, was assessed in vitro and in animal models. The ADSCs were isolated from the buccal fat tissue of six patients using enzymatic digestion and the plastic adherence method. The proliferation and osteogenic differentiation of the cells cultured in XSFM when seeded on the scaffolds were assessed and compared with those of cells cultured in a medium containing fetal bovine serum (FBS). The cell-scaffold constructs were cultured in XSFM and were implanted into calvarial defects in thirty-six Wistar rats to assess new bone regeneration. The proliferation and osteogenic differentiation of the cells in the XSFM medium were notably better than that of the cells in the FBS medium. However, the efficacy of the constructs in enhancing new bone formation in the calvarial defects of rats was not statistically different to that achieved using the scaffolds alone. In conclusion, the PCL-BCP TDP scaffolds were biocompatible and suitable for use as an osteoconductive framework. The XSFM medium could support the proliferation and differentiation of ADSCs in vitro. However, the cell-scaffold constructs had no benefit in the enhancement of new bone formation in animal models.

Estrogen replacement improves skeletal muscle performance by increasing parvalbumin levels in ovariectomized rats.

Muscle weakness is common during menopause. Effective estrogen replacement was hypothesized to prevent sarcopenia. This study aimed to investigate the estrogen level, estrogen receptors (α and ß) immunoreactivities, muscle mass and functions, and parvalbumin (PV) levels in the extensor digitorum longus (EDL) and the gastrocnemius muscles of ovariectomized rats. Adult female Wistar rats (12 weeks old) were divided into five groups: sham-operated (SHAM), and ovariectomized (E0) groups that received 10 weeks of estrogen replacements of 0µg/kg (E0), 10µg/kg (E10), 20µg/kg (E20) and 40µg/kg (E40). The estrogen levels, ER α and ER ß immunoreactivities, muscle fiber sizes and contractivities and the PV levels were reduced in the E0 group, but increased in all the estrogen replacement groups in both muscles. This study indicated that the reduction of estrogen levels led to a decrease of both ER α and ER ß resulting in a decline in muscle mass and PV levels. The decrease of PV levels affected muscle performance, whereas estrogen replacement increased both the ER α and ER ß. The increase in the PV levels may result in an improvement of muscle performance. This may explain one mechanism of estrogen on muscle mass and strength in estrogen dependent sarcopenia.

Quantitative expression of bone-related cytokines induced by mechanical tension-stress during distraction osteogenesis in a rabbit mandible.

AIM: The aim of the present study was to investigate the temporal and spatial gene expression of bone morphogenetic proteins (BMP)-2, -4, and -7, and transforming growth factor-ß (TGF-ß), during the distraction process of the rabbit mandible. METHODS: Twenty rabbits each had an osteotomy on the left mandibular body, and distraction devices were fixed. After a delay of 3 days, distraction was started at a rate of 0.5 mm/12 h for 10 days, followed by a 3-week consolidation phase. Four rabbits were killed 5 and 10 days of distraction, and 1, 2, and 3 weeks after the completion of distraction. The clinical, histological, and radiographic appearances were evaluated and analyzed with the concomitant BMP expression pattern at each interval. RESULTS: After the distraction was started, the fibrous interzone developed between the osteotomy fragments, where intramembranous ossification was noted. The quantitative expression of BMP-2, -4, and -7, and TGF-ß, were increased immediately after active distraction before a gradual decline to normal levels after the third week of consolidation. CONCLUSION: These results suggest that BMP and TGF-ß play an important role in the induction of bone formation during distraction osteogenesis. The selective expression of each bone-related cytokine could provide useful insight into accelerated bone maturation and the treatment of poorly-healing fractures in clinical cases.

The primacy of platelet-rich fibrin on bone regeneration of various grafts in rabbit's calvarial defects.

AIMS: This study investigated the effect of platelet-rich fibrin (PRF) on bone regeneration of various grafting materials in rabbit calvarial defects. MATERIAL AND METHODS: Two bicortical skull defects were prepared in 20 New Zealand white rabbits; 10 rabbits were treated with PRF and the other 10 were non-PRF. In both groups, autogenous bone was compare to empty defects in 5 rabbits and the composite of autogenous bone and deproteinized bovine bone versus deproteinized bovine bone (DBB) in the other five. The animals were sacrificed at 8 weeks. Bone formation was assessed by radiographic densitometry and histomorphometric analysis. RESULTS: The mean optical density (OD) and histomorphometric analysis (HA) of the percentage of new bone showed that the PRF groups were significantly higher than the non-PRF groups in the autogenous bone graft (OD: 0.60 ± 0.19 vs 0.36 ± 0.03; HA: 38.03 ± 4.23 vs 26.21 ± 10.58) and the empty defect (OD: 0.29 ± 0.06 vs 0.11 ± 0.06; HA: 18.81 ± 9.27 vs 6.24 ± 5.01), but not in the DBB group (OD: 1.18 ± 0.17 vs 1.07 ± 0.05; HA: 13.067 ± 3.64 vs 9.63 ± 5.47) and the composite group (OD: 0.81 ± 0.15 vs 0.91 ± 0.05; HA: 22.63 ± 3.61 vs 21.29 ± 3.52). CONCLUSIONS: PRF had a positive effect on bone formation when used alone or combined with autogenous bone, but not with deproteinized bovine bone.

Effects of estrogen via estrogen receptors on parvalbumin levels in cardiac myocytes of ovariectomized rats.

The study investigated the effects of estrogen on parvalbumin (PV) levels in cardiac myocytes of ovariectomized rats, which is a model system for postmenopausal woman. Parvalbumin acts as a relaxing factor in cardiac myocytes. Adult female Wistar rats, 12 weeks old, were randomly divided into 5 groups of 10: sham-operated (SHAM), ovariectomized (OVX), and OVX receiving estrogen replacement of 10 µg/kg (Es10), 20 µg/kg (Es20) and 40 µg/kg (Es40). After 10 weeks, serum estrogen levels were measured and the α and ß estrogen receptors in cardiac myocytes were investigated by immunohistochemistry. PV levels were examined by immunohistochemistry and Western blot analysis. Cardiac myocytes of all animals showed strong staining intensities for α immunoreactive (Es α-ir), but weak staining for ß immunoreactive (Es ß-ir) estrogen receptors. The Es α-ir was reduced in the cardiac myocytes of the OVX groups, but increased in the Es10, Es20 and Es40 groups. We therefore suggest that estrogen effects are mediated via Es α receptors rather than Es ß receptors in female rat hearts. Estrogen and PV immunoreactive (PV-ir) levels and the intensity of the PV band observed in the OVX group were less than those of the SHAM group. In the Es10, Es20 and Es40 groups, the increased intensity of the PV-ir and PV bands correlated with the increased estrogen levels. The low PV levels in cardiac myocytes induced by low estrogen were restored by estrogen replacement therapy. Therefore a reduction of PV may lead to diastolic dysfunction in menopause.

Effects of alcohol administration during adulthood on parvalbumin and glial fibrillary acidic protein immunoreactivity in the rat cerebral cortex.

The pathology of brain atrophy mediated by alcohol was investigated in all parts of the cerebral cortex (the frontal, parietal, temporal lobes and occipital cortex) by using two markers: parvalbumin (PV) and glial fibrillary acidic protein (GFAP). Three-month old male Wistar rats were divided into control (C) and alcohol-exposed groups. The control group received distilled water, whereas the alcohol-exposed groups received either a low dose (2g/kg body wt) or a high dose (5g/kg) of ethanol for periods of 21 days, 3 or 6 months. The brains of the animals were processed for immunohistochemistry using anti-parvalbumin and anti-GFAP antibodies and the number of PV immunoreactive (PV-ir) neurons and GFAP immunoreactive (GFAP-ir) astrocytes were counted per unit area. Results showed that all groups exposed to ethanol had significantly reduced numbers of PV-ir neurons in all parts of the cerebral cortex compared to those of the control group (p<0.05). In contrast, the numbers of GFAP-ir astrocytes were increased in all parts of the cerebral cortex following the exposure to a high dose of ethanol after 21-days (but not a low dose) and both high and low doses of ethanol after 3-months or 6-months treatment compared to those of age-matched control groups (p<0.05). This indicated that in young rats (21-days), PV-ir neurons in all cerebral cortex areas seemed to be more sensitive to alcohol than GFAP-ir astrocytes. Moreover, the change in densities of both PV-ir neurons and GFAP-ir astrocytes became more apparent after exposure to prolonged and high doses of ethanol. The decrease of PV-ir neurons and the increase of GFAP-ir astrocytes indicated that alcohol may induce pathology in broad areas of the cerebral cortex. This may explain the underlying mechanism of brain atrophy and other impairments found in alcoholics. For investigations of the effects of alcohol on mediating brain pathology, we recommend the use of the two markers (PV and GFAP).

Effects of alcohol on the levels of parvalbumin in rat hearts.

Chronic excessive alcohol administration has been reported to be associated with diastolic dysfunction. Parvalbumin (PV) is a calcium-binding protein present in cardiac myocytes and involved in mediating relaxation. Therefore, alteration of PV levels may affect relaxation in cardiac myocytes. This study investigated the effects of alcohol administration on the levels of PV in the rat heart. Male Wistar rats weighing 200-250 g were divided into 2 groups: control (C) and alcohol-treated groups. The control group was provided with distilled water and the alcohol groups were provided with either a low dose (LD, 2g/kg) or high dose of ethanol (HD, 5 g/kg) once daily for 21 days, 3 months or 6 months. The PV levels in the ventricles were investigated by immunohistochemistry and Western blot analysis. In the 21-day ethanol-treated groups, parvalbumin immunoreactivity (PV-ir) and protein levels were not different when compared to the C, LD and HD groups. In the 3-month ethanol-treated groups, PV-ir and PV protein levels were decreased in both the LD and HD groups compared to that of the control group. In the 6-month ethanol-treated groups, PV-ir and PV protein levels decreased significantly in both the LD and HD groups (P<0.05). This indicates that short-term ethanol treatment may not affect PV levels, whereas, long-term ethanol treatment clearly reduced PV levels. The decrease of PV was predominantly due to the direct toxic effects of alcohol rather than malabsorption caused by pathological changes in the duodenum and liver. The toxic effects of alcohol leading to a reduction of PV levels may lead to diastolic impairment.

Alcohol administration during adulthood induces alterations of parvalbumin and glial fibrillary acidic protein immunoreactivity in rat hippocampus and cingulate cortex.

Alcohol induces impairment of cognition, learning and memory. Neurotoxic effects of alcohol on the pathology of the hippocampus and the cingulate cortex were investigated in experimental rats. Parvalbumin (PV), a calcium-binding protein, is a crucial component of GABAergic neurons and glial fibrillary acidic protein immunoreactive (GFAP-ir) astrocytes have been used as markers. We investigated the effects of ethanol exposure during adulthood on the PV-ir neurons and GFAP-ir astrocytes in the hippocampus and the cingulate cortex of 3-month-old male Wistar rats. The rats were divided into 2 groups: control (C) and alcohol-exposed groups. The control group received distilled water whereas the alcohol-exposed groups received either a low dose (20%w/v, LD) or high dose (40%w/v, HD) of ethanol for periods of 21 days, 3 or 6 months. The brains of the animals were processed for immunohistochemistry using anti-parvalbumin and anti-GFAP antibodies and the numbers of PV immunoreactive (PV-ir) neurons and GFAP-ir astrocytes were counted/unit area. For each period of administration, the number of PV-ir neurons was significantly reduced for groups exposed to both the low and the high doses of ethanol compared to those of control groups in both the hippocampus and the cingulate cortex (p<0.01). In addition, the number of PV-ir neurons was progressively reduced after prolonged ethanol exposure. In contrast, there was a significantly increased number of GFAP-ir astrocytes observed in the hippocampus and the cingulate cortex in all groups exposed to ethanol and this was a function of both the duration and the dose of ethanol exposure, indicating that PV-ir neurons are as sensitive as the GFAP-ir astrocytes to ethanol exposure. Our data indicate that alcohol exposure induced a reduction of PV-ir neurons and an increase of GFAP-ir astrocytes in the hippocampus and the cingulate cortex and this may be associated with the impairment of cognition, learning and memory after chronic alcohol administration.

Age-related changes in parvalbumin in the heart of female rats.

Changes of parvalbumin protein levels and immunolocalisation during the postnatal development of the female rat heart were investigated in order to determine if they were correlated with age-related changes in cardiac function. Hearts from newborn, 3-month-old (young), 6-month-old (young adult) and 12-month-old (adult) female Wistar rats were processed for immunohistochemical localization of parvalbumin and for Western blotting assay. Parvalbumin was detected by both methods in all age groups from newborn to 12-month-old rats. In the newborn rat heart, parvalbumin immunoreactivity did not fully fill the sarcoplasm of the cardiac myocytes and the amount of parvalbumin was low compared to the adult levels. In contrast, in 3-12-month-old rats, strong parvalbumin immunoreactivity was detected throughout the sarcoplasm of all cardiac myocytes and the amount of parvalbumin increased with increasing age (from newborn to adult). Our study indicates that an increase of parvalbumin levels in the female rat heart with increasing age may be associated with maintenance of proper relaxation of the cardiac myocytes needed to cope with the increasing workload of the heart during postnatal growth.

Influence of aging and long-term swimming exercise on parvalbumin distribution in rat hearts.

Parvalbumin (PV), which is a small (12kDa) cytoplasmic calcium-binding protein, has been implicated in mediating relaxation in cardiac myocytes. The influence of aging and exercise on the distribution of PV in rat heart was investigated. Male Wistar rats aged 3, 6, 12 and 18-months were divided into sedentary and exercise groups. The exercise group underwent exercise in the form of regular swimming for 6 months. The hearts were processed for immunohistochemistry and Western blotting. The intensity of PV immunoreactivity was strong in the 9 and 12-month hearts and decreased in the 18-month hearts. The smallest amount was detected in the 24-month rat hearts when compared to those of the 9, 12 and 18-month rat hearts. Significantly less PV was detected in the 18 and 24-month hearts compared to the 12-month rat hearts (P<0.05). The intensity of PV immunoreactivity was considerably stronger in hearts of the 9, 12 and 18-months exercised rats than in hearts of age-matched sedentary rats. However, in the hearts of 24-month rats, immunoreactivity was only slightly stronger in the exercised rats in comparison with those of sedentary rats. A significant increase of PV detection in hearts was found in the exercised rats in comparison with sedentary rats in the 9 (P<0.05) and 18-month samples (P<0.01). Our data indicate that PV is down-regulated in the rat heart during aging. In addition, our data indicate that long-term swimming exercise could induce an increase of PV expression.

Immunohistochemical localization of parvalbumin calcium-binding protein in the heart tissues of various species.

Parvalbumin (PV), a cytoplasmic high-affinity Ca(2+)-binding protein, was recently identified in rat heart tissue and has been implicated in mediating relaxation in cardiac myocytes. The presence of PV in the heart of mouse, chicken, rabbit and pig was studied using immunohistochemistry. PV immunoreactivity (PV-ir) was identified in the heart of all four species. All cardiac myocytes of each species had an identical pattern of PV-ir in their cytoplasm. The highest intensity of PV-ir was observed in mouse and chicken cardiac myocytes. The intensity of PV-ir was lower in rabbit cardiac myocytes and lowest in pig cardiac myocytes compared to those of chicken and mouse. PV-ir was observed in the walls of all four cardiac chambers (left and right atria and left and right ventricles), with the left ventricle, in general, having the highest labeling intensity. The intensity of PV-ir may be correlated with the physical activity of the heart of each species. Some potential applications of these data for treatment of human diastolic heart dysfunctions are discussed.

Osteoconductive effects of 3 heat-treated hydroxyapatites in rabbit calvarial defects.

PURPOSE: This study aimed to detect the osteoconductive ability of 3 bovine hydroxyapatites (HAs) that were sintered at 800 degrees C (HA800), 1,200 degrees C (HA1200), and 1,350 degrees C (HA1350), according to new bone formation. MATERIAL AND METHODS: Two bicortical skull defects were prepared in 10 New Zealand white rabbits. Four rabbits were used as controls; in each, 1 defect was filled with autogenous bone chips and the contralateral defect was left empty for the critical size defect (CSD). The other 6 rabbits had a total of 12 defects, 4 each randomly filled with HA 800, HA1200, or HA1350. The animals were sacrificed at 8 weeks. New bone formation was assessed by radiographic densitometry and histomorphometric analysis. RESULTS: The mean optical density (OD) of the CSD group (0.092 +/- 0.006) was less than that of the autogenous bone chip (0.102 +/- 0.002), HA1200 (0.108 +/- 0.005), and HA 1350 (0.102 +/- 0.003) groups. The mean OD of the HA 1200 group was significantly different from that of the HA 800 group (0.094 +/- 0.003). The histomorphometric analysis demonstrated that the autogenous bone chip group (34.89 +/- 4.61) was significantly different from the CSD (12.16+/- 6.97), HA 800 (18.32 +/- 7.33), and HA1350 (13.99 +/- 3.94) groups, but not the HA1200 group (24.83 +/- 12.12). CONCLUSIONS: The findings demonstrate that heat-treated bovine HA enhances bone formation, and HA 1200 tends to provide greater bone formation than the other 2 HAs.

Up-regulation of parvalbumin expression in newborn and adult rat heart.

Parvalbumin (PV), a cytoplasmic calcium-binding protein, functions as a relaxing factor and has recently been detected in rat heart. Developmental changes in PV localization and expression were investigated in the heart of Wistar rats at different ages. Ten hearts from newborn, 3-month-old (young), 6-month-old (young adult), and 12-month-old (adult) rats were processed for immunohistochemistry and Western blot assay. PV was detected in hearts of all the age groups of the rats from newborn to 12-month-old by both immunohistochemistry and Western blotting. A variable distribution of PV immunoreactivity was present in newborn cardiac myocytes. In the 3-, 6-, and 12-month-old rat hearts, identical PV immunoreactivity was found in all cardiac myocytes and the intensity of PV immunoreactivity increased with increasing age. By using Western blotting, it was found that the expression of PV was low in the newborn rat heart and increased with increasing age. The presence of PV may correlate with the physiological age, and possibly serves to maintain proper relaxation of the cardiac myocytes to cope with an increasing workload of the heart during body growth.

Angiotensin II may mediate apoptosis via AT1-receptors in the rat cardiac conduction system.

INTRODUCTION: Apoptosis and angiotensin II (Ang II) have been suggested as possible causes of arrhythmias. In addition, Ang II via Ang II type I (AT(1)-) receptors, has been demonstrated to induce cardiomyocyte apoptosis. The transgenic m(Ren-2)27 (TG) rat carries the additional Ren-2 gene, the expression of which results in an increase in cardiac Ang II, thus potentially affecting the cell growth/death equilibrium. In this study we have investigated the effect of Ang II, via AT(1)-receptors, on mediating apoptosis in a cardiac conduction system (SA node and AV nodes). MATERIALS AND METHODS: Heart sections from male two-day, one-week and two-week TG and Sprague-Dawley (SD) rats were stained with Masson Trichrome to localise the SA and AV nodes. The sections containing SA or AV nodes were processed for quantitation of apoptotic nuclei and AT(1)-receptors. RESULTS: The number of apoptotic nuclei/mm(2) in the SA and AV nodes were found to decrease from two days to two weeks in both the TG and the SD rats, and the number of apoptotic nuclei/mm(2) in the TG groups was significantly higher than that of the SD groups for all ages (p<0.05). The number of AT(1)-receptors/mm(2) in the SA node were found to decrease with increasing age, whereas the number of AT(1)-receptors/mm(2) in the AV node was increased in both TG and SD rats and the number of AT(1)-receptors/mm(2) in the three TG groups was significantly more than that of the three SD groups (p<0.05). DISCUSSION AND CONCLUSION: As a consequence of the additional renin gene in the TG rats, which results in the alteration of the local renin-angiotensin system, the numbers of AT(1)-receptors/mm(2) and apoptotic nuclei/mm(2) are increased. The number of apoptotic nuclei/mm(2) and AT(1)-receptors/mm(2) in the SA node decrease with maturation, whereas, the number of AT(1)-receptors in the AV node increase. Thus, there may be a correlation between Ang II and apoptosis in the SA node, which does not appear to be present in the AV node.