Eggshells as natural calcium carbonate source in combination with hyaluronan as beneficial additives for bone graft materials, an in vitro study.

INTRODUCTION: In bone metabolism and the formation especially in bone substitution, calcium as basic module is of high importance. Different studies have shown that the use of eggshells as a bone substitute material is a promising and inexpensive alternative. In this in vitro study, the effects of eggshell granulate and calcium carbonate towards primary bovine osteoblasts were investigated. Hyaluronan (HA) was used as artificial extracellular matrix (ECM) for the used cells to facilitate proliferation and differentiation and to mimic the physiological requirements given by the egg in vivo. METHODS: Hyaluronan, eggshells, a combination of hyaluronan and eggshells and CaCO3 were applied to the cells as additive to the used standard medium (modified High Growth Enhancement Medium) in a concentration of 0,1 g/l. The effect of the additives in the culture medium was examined by proliferation tests, immunohistochemical staining (anti-collagen type I, anti-osteopontin, anti-osteonectin and anti-osteocalcin) and kinetic oxygen measurements. RESULTS: Our investigations revealed that all investigated additives show beneficial effect on osteoblast activity. Cell proliferation, differentiation and the metabolic activity of the differentiated cells could be influenced positively. Especially in the case cell cultures treated with eggshells the strongest effects were detected, while for the hyaluronan compared with eggshells, a weaker increase in cell activity was observed. CONCLUSION: In summary, it can be stated that the investigated components come into consideration as beneficial supplements for bone graft materials especially for maxillo facial surgery application.

In vitro inhibition of HUVECs by low dose methotrexate - insights into oral adverse events.

BACKGROUND: With socio-economic changes, dentists and maxillofacial surgeons are more and more faced with medically compromised patients. Especially, the admission of antirheumatic drugs has increased remarkably. So dentists and maxillofacial surgeons should be aware of related adverse reactions that affect the craniofacial region. To identify possible cellular effects of disease modifying antirheumatic drugs (DMARDs) we investigated the influence of methotrexate (MTX) on human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were incubated with various concentrations of MTX, corresponding to serum concentrations found in rheumatoid arthritis (RA) patients. The effect of MTX on cell proliferation, differentiation as well as mitochondrial activity was measured by use of immunostaining, cell counting and 3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: All samples incubated with MTX (1-1000 nM) showed significantly decreased cell viability when compared to controls. Cells were less proliferating, but did not lose their ability to synthesize endothelial proteins. A slight dose dependency of inhibiting effects was demonstrated. The observed differences between control and sample groups were rising with longer duration. CONCLUSION: Because of the crucial role of endothelial cells and their precursor cells in wound healing, a negative influence of MTX on oral health has to be supposed, correlating to clinical observations of adverse reactions in the oral cavity, such as ulcerative or erosive lesions.

Quantitative analysis of dynamic behavior of osteoblasts during in vitro formation of micro-mass cell cultures.

Improvements in bone tissue engineering require an understanding of cellular and tissue level behavior of osteoblast-like cells. Experiments indicate that in the absence of an anchoring material, intercellular adhesion may be based on signals that promote cell activity resulting in the formation of a spheroid cell-matrix. The aim of the present study is to investigate the formation of scaffold-free three-dimensional micro-mass cell spheroids in vitro, and to characterize quantitatively the cell movement. A new correlation based automated tracking method is evaluated in order to optimize the processing parameters and to identify statistical parameters that characterize the cell behavior. Results suggest that the temporal development of the mean distance of the cells to the center of gravity may be described by an exponential function, thus providing a characteristic time constant as a quantitative measure of cell dynamics. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

An in vitro study of osteoblast vitality influenced by the vitamins C and E.

Vitamin C and vitamin E are known as important cellular antioxidants and are involved in several other non-antioxidant processes. Generally vitamin C and vitamin E are not synthesized by humans and therefore have to be applied by nutrition. The absence or deficiency of the vitamins can lead to several dysfunctions and even diseases (e.g. scurvy). The main interest in this study is that vitamin C and E are known to influence bone formation, e.g. vitamin C plays the key role in the synthesis of collagen, the major component of the extracellular bone matrix.In the present study we evaluate the effect of ascorbic acid (vitamin C) and α-tocopherol (vitamin E) on the proliferation and differentiation of primary bovine osteoblasts in vitro. Starting from standard growth medium we minimized the foetal calf serum to reduce their stimulatory effect on proliferation.An improved growth and an increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin was observed while increasing the ascorbic acid concentration up to 200 µg/ml. Furthermore the effects of α-tocopherol on cell growth and cell differentiation were examined, whereby neither improved growth nor increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin were detected.Further investigations are necessary to target at better supportive effect of vitamins on bone regeneration, and healing.

Impact of testosterone on the expression of organic anion transporting polypeptides (OATP-1A2, OATP-2B1, OATP-3A1) in malignant and non-malignant human breast cells in vitro.

OBJECTIVES: Postmenopausal hormone therapy (HT) increases local estrogen formation in breast tissue. The enzymatic substrates depend on transmembrane anion transporting polypeptides (OATPs) to reach intracellular enzymes. The aim of this study was to investigate the effect of testosterone (T) on the expression of OATP-1A2, OATP-2B1, and OATP-3A1 in malignant (MCF-7, BT-474) and non-malignant (HBL-100) breast cells in vitro. STUDY DESIGN: Cells were incubated in RPMI 1640 medium containing 5% steroid-depleted fetal calf serum for 3d, and subsequently incubated in the absence or presence of T, anastrozole (A), and T+A (10(-6)M) for 24h at 37°C. MAIN OUTCOME MEASURES: OATP expression was determined by immunocytochemical staining. Expression intensity was graded as low, moderate, or strong. Hormone receptor (AR, PR, ESR1, ESR2) expression was investigated by qPCR and Western blotting. Rank variance analysis was performed for statistical analysis (p≤0.05). RESULTS: OATP-1A2, OATP-2B1, and OATP-3A1 expression was present in all untreated breast cell lines examined, with OATP-1A2 and OATP-3A1 being the predominant ones. There was a trend for a higher baseline expression in untreated HBL-100 and BT-474 in comparison to MCF-7 cells, which was significant for OATP-2B1. T treatment led to decreased OATP-1A2, -2B1, and -3A1 expression in BT-474 and HBL-100 cells, respectively. In contrast, in MCF-7 cells, OATP-2B1 expression was significantly increased. T-induced upregulation of AR and PR protein expression in BT-474 and MCF-7 cells was reduced by A treatment. CONCLUSIONS: T may constitute a signal for differential regulation of mammary OATP expression. In non-malignant breast cells T seems to have a beneficial effect by reducing the availability of substrates for the intracellular formation of potent estrogens.

Decrease in the expression of the type 1 PTH/PTHrP receptor (PTH1R) on chondrocytes in animals with osteoarthritis.

BACKGROUND: To evaluate the expression of the type 1 PTH/PTHrP receptor (PTH1R) on chondrocytes from hyaline cartilage over the course of osteoarthritis (OA). METHODS: In 12 NZW rabbits, the anterior cruciate ligament (ACL) was resected to create anterior instability of the knee. In 12 control rabbits, only a sham operation, without resection of the ACL, was performed. Four animals from each group were killed at 3, 6, and 12 weeks. After opening the knee joint, OA was macroscopically graded and hyaline cartilage of the load-bearing area was evaluated histologically according to the Mankin scale and by immunostaining for PTH1R. RESULTS: There was a positive linear correlation between the time after surgery and the macroscopic and histologic OA scores. The scores in the control group were constant over the time course. Immunostaining showed significantly less expression of PTH1R in the experimental compared to the control group after 6 (P < 0.05) and 12 weeks (P < 0.01). In the experimental group, a negative linear correlation between PTH1R expression and macroscopic and histologic grades was found. CONCLUSIONS: The results show an in vivo decrease in the expression of PTH1R on chondrocytes over the time course of OA. Further studies are needed to evaluate whether new treatment approaches could evolve from this knowledge.

Effects of hormone therapy on estrogen synthesis from E1S in the mammary gland of postmenopausal women.

OBJECTIVES: To investigate the effect of hormone therapy (HT) on the expression of hormone receptors and the ex vivo estrogen biosynthesis in the breast. METHODS: Comparative studies were carried out in breast tissue from 28 postmenopausal women undergoing breast surgery due to breast cancer (BC). Glandular breast tissue at least 1cm distant from the tumor was analyzed. Groups included patients having received HT (n=18), and non-user of HT (controls, n=10) prior to BC diagnosis. Steroid sulfatase (STS) activity was evaluated by incubating homogenized breast tissue with [3H]-estrone sulfate. Thin layer chromatography was performed to separate the products estrone (E1) and estradiol (E2). Histomorphometry for breast tissue composition and immunohistochemistry for expression of estrogen receptor (ER) alpha and beta as well as progesterone receptor (PR) were performed. RESULTS: In all groups, significantly more E2 than E1 was produced. Local E2 formation was higher in women having been treated with estrogen and progestogen (p< or =0.05). Local EZ formation was positively correlated with ER alpha expression (r(s)=0.5; p=0.03). Histomorphometrical and immunohistochemical outcomes did not differ between groups. However, the amount of vessels was higher in women having been treated with HT compared to controls (p=0.09). CONCLUSIONS: Long-term HT increases local estrogen formation in normal human breast tissue.

Deer antler regeneration: cells, concepts, and controversies.

The periodic replacement of antlers is an exceptional regenerative process in mammals, which in general are unable to regenerate complete body appendages. Antler regeneration has traditionally been viewed as an epimorphic process closely resembling limb regeneration in urodele amphibians, and the terminology of the latter process has also been applied to antler regeneration. More recent studies, however, showed that, unlike urodele limb regeneration, antler regeneration does not involve cell dedifferentiation and the formation of a blastema from these dedifferentiated cells. Rather, these studies suggest that antler regeneration is a stem-cell-based process that depends on the periodic activation of, presumably neural-crest-derived, periosteal stem cells of the distal pedicle. The evidence for this hypothesis is reviewed and as a result, a new concept of antler regeneration as a process of stem-cell-based epimorphic regeneration is proposed that does not involve cell dedifferentiation or transdifferentiation. Antler regeneration illustrates that extensive appendage regeneration in a postnatal mammal can be achieved by a developmental process that differs in several fundamental aspects from limb regeneration in urodeles.

Cellularity and apoptosis after radiofrequency-induced shrinkage of collagenous tissue: assessment of postoperative immobilization using an in vivo rabbit model.

BACKGROUND: Electrothermally-assisted capsular shrinkage has been gaining increased acceptance in the treatment of shoulder instability. Its indication in ACL-deficient knees has been discussed recently. METHODS: We examined the influence of immobilization on cell homeostasis of healing collagenous tissue after radiofrequency energy was applied to the patellar tendon in 23 rabbits. The animals were killed immediately after surgery (n = 6) or 3 weeks after surgery (n = 17). 10 rabbits were allowed normal cage activity, whereas the treated hind limb of 7 animals was immobilized for 3 weeks in a cast. Feulgen staining was used to stain the DNA of cell nuclei. Cells undergoing apoptosis were identified by the TUNEL method. Quantitative histological assessment was performed using imaging analysis software. RESULTS: Severe cellular damage in RF-treated collagenous tissue was partly induced by the immediate onset of apoptosis. At 3 weeks after surgery, non-immobilized tendon showed increased cellularity and apoptosis, whereas immobilization prevented the increase in cellularity and apoptosis significantly. The calculated ratio of apoptosis was not influenced by any postoperative treatment. INTERPRETATION: Diminished cellularity and apoptosis during tissue remodeling, due to immobilization, may protect the shortened collagenous scaffold from stretching and further optimize the clinical outcome after radiofrequency shrinkage. To stabilize the shrunken tissue, proliferation during postoperative wound healing should be minimized by careful rehabilitation.

Increase in the expression of the transmembrane surface receptor CD44v6 on chondrocytes in animals with osteoarthritis.

OBJECTIVE: To evaluate the expression of CD44v6 on chondrocytes from hyaline cartilage over the course of osteoarthritis (OA). METHODS: In 12 NZW rabbits, the anterior cruciate ligament (ACL) was resected to create anterior instability of the knee. In 12 control rabbits, only a sham operation, without resection of the ACL, was performed. Four animals from each group were killed at 3, 6, and 12 weeks. After opening the knee joint, OA was macroscopically graded and hyaline cartilage of the load-bearing area was evaluated histologically according to the Mankin scale and by immunostaining for CD44v6. RESULTS: There was a positive linear correlation between the time after surgery and the macroscopic and histologic OA scores. The scores in the control group were constant over the time course. Immunostaining showed constant expression of CD44v6 in the control group. In the experimental group, a positive linear correlation between CD44v6 expression and macroscopic and histologic grades was found. CONCLUSION: The results show an in vivo increase in the expression of the hyaluronan receptor CD44 over the time course of OA. Further studies are needed to evaluate whether this pattern applies to humans and whether new treatment approaches could evolve from this knowledge.

Image-based extracorporeal tissue engineering of individualized bone constructs.

PURPOSE: Computer-aided technologies have been recently employed for use in extracorporeal bone tissue engineering strategies. In this pilot animal experimental study, the intention was to test whether autologous osteoblast-like cells cultured in vitro on individualized scaffolds can be used to support bone regeneration in a clinical environment. MATERIALS AND METHODS: For this purpose, mandibular bone defects were surgically introduced into the mandibles of minipigs and the scaffold of the defect site was modeled by computer-aided design/computer-aided manufacturing technique. Autologous bone cells from porcine calvaria were harvested from minipigs and grown in culture. Cells were seeded on scaffolds generated by rapid prototyping of polylactic acid/polyglycolic acid copolymers. The defects were then reconstructed by implanting the tissue constructs. RESULTS: The intraoperative sites as well as the postoperative computerized tomographic scans demonstrated an accurate fit in the defect sites. The implanted scaffold constructs enriched with osteoblast-like cells were well tolerated and appeared to support bone formation, as revealed by histologic and immunohistochemical analyses. DISCUSSION: These results indicated that in vitro expanded osteoblast-like cells spread on a resorbable individualized scaffold can be capable of promoting the repair of bony defects in vivo. CONCLUSION: These results warrant further attempts to combine computer modeling and tissue engineering for use in bone reconstructive surgery.

Bone tissue engineering by primary osteoblast-like cells in a monolayer system and 3-dimensional collagen gel.

PURPOSE: To engineer living bone tissue in vitro, bone cells must be multiplied and differentiated in cell culture. Osteoblasts are known to be the crucial cells responsible for the bone modeling process. Periosteal-derived osteoblasts were therefore cultured for up to 3 weeks in Petri dishes as well as in a 3-dimensional collagen gel. METHODS: Proliferation, migration, and differentiation of cells as well as the synthesis of extracellular matrix proteins were monitored during the culture period by histology, electron microscopy, and immunohistochemistry. Mineral formation was investigated by electron diffraction studies and element analysis. RESULTS: Osteoblasts proliferated and migrated in Petri dishes as well as in the collagen gel without loss of viability during the whole experimental period. They demonstrated a mature osteoblast phenotype as indicated by the synthesis of a bone-like extracellular matrix. They formed an extracellular matrix containing osteocalcin, osteonectin, and newly synthesized collagen type I in both environments. Mineral formation was seen in colocalization with the bone-like extracellular matrix proteins in Petri dishes. Microanalytical investigations revealed a matrix vesicle-mediated mineral formation at early stages of culture. CONCLUSIONS: Our cell culture confirmed the ability to multiplicate differentiated and viable osteoblast-like cells in 2- and 3-dimensional space. Additionally, bone-like mineralization can be induced by primary osteoblasts in monolayer culture. The data suggest that this approach can be used as a tool in bone tissue engineering.

Histological studies of bone formation during pedicle restoration and early antler regeneration in roe deer and fallow deer.

The purpose of the present study was to examine the process of bone formation in the regenerating cranial appendages of roe deer (Capreolus capreolus) and fallow deer (Dama dama) during the early postcasting period. After the antlers are cast, osteoclastic and osteoblastic activities lead to a smoothing of the pedicle's separation surface, a strengthening of the pedicle bone, and a partial restoration of the distal pedicle portion that was lost along with the cast antler. Initially, bone formation occurs by intramembranous ossification, but early during the regeneration process cartilage is formed at the tips of the cranial appendages, and is subsequently replaced by bone in a process of endochodral ossification. Shortly after the antlers are cast, the cambium layer of the periosteum in the distal pedicle is markedly enlarged, which suggests that the periosteum serves as a cell source for the bone-forming tissue covering the exposed pedicle bone. The histological findings of our study are consistent with the view that the bony component of the regenerating cranial appendages of deer is largely derived from the pedicle periosteum. Based on findings in other bone systems, we speculate that stem cells that can undergo both osteogenic and chondrogenic differentiation are present in the pedicle periosteum. The early onset of chondrogenesis in the regeneration process is regarded as an adaptation to the necessity of producing a huge volume of bone within a short period. This parallels the situation in other cases of chondrogenesis in membrane bones.

Histochemical and ultrastructural studies of cartilage resorption and acid phosphatase activity during antler growth in fallow deer (Dama dama).

Cartilage resorption in forming primary fallow deer antlers was studied by histochemistry and electron microscopy. A high activity of tartrate-resistant acid phosphatase (TRAP), a histochemical marker of skeletal resorbing cells, was first detected in cells located in the mesenchymal tissue separating the columns of hypertrophic cartilage. No cartilage resorption was observed in this region. Intense TRAP staining occurred in large multinucleated cells (identified as inactive osteoclasts) as well as in smaller cells (regarded as mononuclear osteoclast progenitors). On the basis of these findings it was concluded that this was the region where osteoclasts differentiated from progenitor cells. Further proximally, the mineralized cartilage was eroded by active osteoclasts that were located in Howship's lacunae and exhibited an intense TRAP staining. Electron microscopy showed that the cells identified as inactive osteoclasts lacked a polarized organization. In contrast, the active osteoclasts in the zone of cartilage resorption exhibited a typical polarized organization: the nuclei congregated near the basolateral cell surface, and there was a zone of deep membrane infoldings (ruffled border) surrounded by a clear zone at the apical cell pole adjacent to the resorption surface of the mineralized cartilage. The multinucleated cartilage-resorbing cells of the forming antler thus exhibited the typical histochemical and morphological features of active mammalian osteoclasts. Low levels of TRAP activity were also observed in hypertrophic chondrocytes; however, the specificity and potential significance of this staining remain to be elucidated.