Osteoblastic Tissue Research Articles

A Spontaneous 3D Bone‐On‐a‐Chip for Bone Metastasis Study of Breast Cancer Cells

Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone-on-a-chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co-culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone-on-a-chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis.

In Vitro Mimics of Bone Remodeling and the Vicious Cycle of Cancer in Bone

Bone remodeling is a natural process that enables growth and maintenance of the skeleton. It involves the deposition of mineralized matrix by osteoblasts and resorption by osteoclasts. Several cancers that metastasize to bone negatively perturb the remodeling process through a series of interactions with osteoclasts, and osteoblasts. These interactions have been described as the "vicious cycle" of cancer metastasis in bone. Due to the inaccessibility of the skeletal tissue, it is difficult to study this system in vivo. In contrast, standard tissue culture lacks sufficient complexity. We have developed a specialized three-dimensional culture system that permits growth of a non-vascularized, multiple-cell-layer of mineralized osteoblastic tissue from pre-osteoblasts. In this study, the essential properties of bone remodeling were created in vitro by co-culturing the mineralized collagenous osteoblastic tissue with actively resorbing osteoclasts followed by reinfusion with proliferating pre-osteoblasts. Cell-cell and cell-matrix interactions were determined by confocal microscopy as well as by assays for cell specific cytokines and growth factors. Osteoclasts, differentiated in the presence of osteoblasts, led to degradation of the collagen-rich extracellular matrix. Further addition of metastatic breast cancer cells to the co-culture mimicked the vicious cycle; there was a further reduction in osteoblastic tissue thickness, an increase in osteoclastogenesis, chemotaxis of cancer cells to osteoclasts and formation of cancer cells into large colonies. The resulting model system permits detailed study of fundamental osteobiological and osteopathological processes in a manner that will enhance development of therapeutic interventions to skeletal diseases.

A comparative study of pulp vitality following vital pulpotomy using calcium hydroxide-based sealants in beagle dogs

Vital pulpotomy is very useful method for disarming of canine tooth, tooth fracture, periodontitis, and malocclusion in veterinary dentistry. And calcium hydroxide is the commonly used material as liner during vital pulpotomy. This creates a mineralized barrier by stimulating osteoblastic hard tissue repair, arrests the inflammatory response, and soothes dentin. However, the powder or mix type calcium hydroxide materials have many disadvantages because of complicating procedure to use and are hard to be handled when vital pulpotomy is followed under general anesthesia in animals. This study was performed to compare the effect of mix and premixed paste type calcium hydroxide as liner on vital pulpotomy. Six beagle dogs had hemisection on mesial root of mandibular first molar and were performed vital pulpotomy on distal root of first molar. On the distal root of left and right mandibular first molar, mix type (DYCAL ® , Dentsply, USA) and premixed paste type calcium hydroxide (VITAPEX ® , Morita, Japan) were used as liner, respectively. The radiological evaluation performed at immediate, 4, 12 and 20 weeks after vital pulpotomy. In results, all teeth had dentinal bridges formed well, there were no periradicular lucency, lamina dura loss and anomalies of pulp cavity. Following this results, on vital pulpotomy in animals, premixed paste type calcium hydroxide was easy to handle and decrease the anesthesia period by more convenient application procedure. Further study is needed for many clinical cases to inspect side effects and other problems.

Multimodal μCT/μMR based semiautomated segmentation of rat vertebrae affected by mixed osteolytic/osteoblastic metastases

Multimodal microimaging in preclinical models is used to examine the effect of spinal metastases on bony structure; however, the evaluation of tumor burden and its effect on microstructure has thus far been mainly qualitative or semiquantitative. Quantitative analysis of multimodality imaging is a time consuming task, motivating automated methods. As such, this study aimed to develop a low complexity semiautomated multimodal μCT/μMR based approach to segment rat vertebral structure affected by mixed osteolytic/osteoblastic destruction. Mixed vertebral metastases were developed via intracardiac injection of Ace-1 canine prostate cancer cells in three 4-week-old rnu/rnu rats. μCT imaging (for high resolution bone visualization), T1-weighted μMR imaging (for bone registration), and T2-weighted μMR imaging (for osteolytic tumor visualization) were conducted on one L1, three L2, and one L3 vertebrae (excised). One sample (L1-L3) was processed for undecalcified histology and stained with Goldner's trichome. The μCT and μMR images were registered using a 3D rigid registration algorithm with a mutual information metric. The vertebral microarchitecture was segmented from the μCT images using atlas-based demons deformable registration, levelset curvature evolution, and intensity-based thresholding techniques. The μCT based segmentation contours of the whole vertebrae were used to mask the T2-weighted μMR images, from which the osteolytic tumor tissue was segmented (intensity-based thresholding). Accurate registration of μCT and μMRI modalities yielded precise segmentation of whole vertebrae, trabecular centrums, individual trabeculae, and osteolytic tumor tissue. While the algorithm identified the osteoblastic tumor attached to the vertebral pereosteal surfaces, it was limited in segmenting osteoblastic tissue located within the trabecular centrums. This semiautomated segmentation method yielded accurate registration of μCT and μMRI modalities with application to the development of mathematical models analyzing the mechanical stability of metastatically involved vertebrae and in preclinical applications evaluating new and existing treatment effects on tumor burden and skeletal microstructure.

Dynamic interaction between breast cancer cells and osteoblastic tissue: Comparison of Two‐ and Three‐dimensional cultures

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi-cell-layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of "Single cell file" commonly observed in end-stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3-E1 murine osteoblasts and MDA-MB-231 human metastatic breast cancer cells, or the metastasis-suppressed line, MDA-MB-231BRMS1, for comparison. When osteoblasts were co-cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro-inflammatory cytokine IL-6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle-shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis-suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts.

Osteogenesis in vitro: from pre-osteoblasts to osteocytes

Murine calvariae pre-osteoblasts (MC3T3-E1), grown in a novel bioreactor, proliferate into a mineralizing 3D osteoblastic tissue that undergoes progressive phenotypic maturation into osteocyte-like cells. Initially, the cells are closely packed (high cell/matrix ratio), but transform into a more mature phenotype (low cell/matrix ratio) after about 5 mo, a process that recapitulates stages of bone development observed in vivo. The cell morphology concomitantly evolves from spindle-shaped pre-osteoblasts through cobblestone-shaped osteoblasts to stellate-shaped osteocyte-like cells interconnected by many intercellular processes. Gene-expression profiles parallel cell morphological changes, up-to-and-including increased expression of osteocyte-associated genes such as E11, DMP1, and sclerostin. X-ray scattering and infrared spectroscopy of contiguous, square centimeter-scale macroscopic mineral deposits are consistent with bone hydroxyapatite, showing that bioreactor conditions can lead to ossification reminiscent of bone formation. Thus, extended-term osteoblast culture (< or =10 mo) in a bioreactor based on the concept of simultaneous growth and dialysis captures the full continuum of bone development otherwise inaccessible with conventional cell culture, resulting in an in vitro model of osteogenesis and a source of terminally differentiated osteocytes that does not require demineralization of fully formed bone.

Osteosarcoma in a pregnant patient with McCune–Albright syndrome

Malignant transformation of fibrous dysplasia is very rare and has not been previously described in patients with McCune–Albright syndrome in the absence of radiation treatment during gestation. Here, we report a 38-year-old pregnant woman with McCune–Albright syndrome and acromegaly accompanied by osteosarcoma. The patient was in the 6th week of pregnancy, when she visited our hospital. She had multiple fibrous dysplasia, skin pigmentation, and acromegaly. The markedly high bone turnover rate during pregnancy tended to decrease after a normal delivery. Fibrous dysplasia of the lower jaw rapidly increased in the 37th week of pregnancy, and the tumor was surgically resected after delivery. Pathological examination of the resected tumor revealed fibrous dysplasia admixed with osteosarcoma containing chondroblastic and osteoblastic tissue. We firstly reported a case of osteosarcoma in a patient with McCune–Albright syndrome, which rapidly progressed during pregnancy.

A Three-Dimensional Osteogenic Tissue Model for the Study of Metastatic Tumor Cell Interactions with Bone

A specialized bioreactor based on the principle of simultaneous growth and dialysis permits growth of three-dimensional (3D), multiple-cell-layer osteogenic tissue from isolated osteoblasts over long, continuous-culture intervals (tested up to 10 months with no sign of necrosis). The resulting tissue recapitulates the stages of bone development observed in vivo, including phenotypic maturation of cobblestone-shaped osteoblasts into stellate-shaped osteocytes interconnected by many intercellular processes. Gene expression profiles parallel cell-morphologic changes with time, ultimately leading to increased expression of osteocyte-associated molecules such as E11, DMP1, and sclerostin. Contiguous, cm(2)-scale macroscopic mineral deposits that form within the bioreactor are consistent with bone hydroxyapatite. The simple to use bioreactor system provides an in vitro model that permits the study and manipulation of cancer cell interactions with bone tissue in real time. Metastatic human breast cancer cells, MDA-MB-231(GFP), introduced into the model grow and colonize osteoblastic tissue in a manner reflecting various characteristics of pathologic tissue observed in the clinic. Specifically, MDA-MB-231(GFP) cells are observed to penetrate the thick extracellular matrix in which osteoblasts are embedded and to form chains reminiscent of "Indian files," described for infiltrating lobular or metaplastic breast carcinomas. Osteoblasts appear to be marshaled into a parallel alignment with cancer cells, followed by erosion of extracellular matrix structural integrity. Tissue degradation appears to be accompanied by increased expression of osteoblast inflammatory cytokines.

Metastatic breast cancer cells colonize and degrade three-dimensional osteoblastic tissue in vitro

Metastatic breast cancer cells (BCs) colonize a mineralized three-dimensional (3D) osteoblastic tissue (OT) grown from isolated pre-osteoblasts for up to 5 months in a specialized bioreactor. Sequential stages of BC interaction with OT include BC adhesion, penetration, colony formation, and OT reorganization into "Indian files" paralleling BC colonies, heretofore observed only in authentic pathological cancer tissue. BCs permeabilize OT by degrading the extra-cellular collagenous matrix (ECM) in which the osteoblasts are embedded. OT maturity (characterized by culture age and cell phenotype) profoundly affects the patterns of BC colonization. BCs rapidly form colonies on immature OT (higher cell/ECM ratio, osteoblastic phenotype) but fail to completely penetrate OT. By contrast, BCs efficiently penetrate mature OT (lower cell/ECM ratio, osteocytic phenotype) and reorganize OT. BC colonization provokes a strong osteoblast inflammatory response marked by increased expression of the pro-inflammatory cytokine IL-6. Furthermore, BCs inhibit osteoblastic bone formation by down-regulating synthesis of collagen and osteocalcin. Results strongly suggest that breast cancer disrupts the process of osteoblastic bone formation, in addition to upregulating osteoclastic bone resorption as widely reported. These observations may help explain why administration of bisphosphonates to humans with osteolytic metastases slows lesion progression by inhibiting osteoclasts but does not bring about osteoblast-mediated healing.

Mechanical properties of the periosteum in the pig, Sus scrofa

The fibrous periosteum forms an intermediary between muscle and ligament forces and the underlying osteoblastic tissue, thus the mechanical properties of the periosteum are critical to understanding osteogenic stimuli. Regional and directional variation in periosteal properties may contribute to the biomechanical regulation of growth in some bones. Periostea of the pig mandibular body, zygomatic arch and metacarpal were loaded to failure under continuous tension. Each tissue type was tested in both the long-axis and transverse orientation. Stiffness, peak stress and peak strain were compared between orientations and among regions. Within the zygomatic periosteum there was little indication of regional difference, and neither zygomatic nor mandibular periosteum showed directional differences. The metacarpal periosteum showed a directional effect only in peak strain, which was greater longitudinally than transversely. There were striking differences, however, among the periostea of the three bones. The zygomatic arch periosteum was the stiffest tissue (91.7±30.5 MPa) and showed the highest strength (12.3±4.6 MPa). The metacarpal periosteum demonstrated slightly lower stiffness and strength (84.7±35.1 and 11.3±5.3 MPa), and peak strains in zygomatic and metacarpal periostea were similarly high (17.7±3.7 and 17.9±3.7 MPa, respectively). The periosteum of the mandibular body was the most deformable tissue (63.0±25.4 MPa), with the lowest-peak strain (15.6±3.0 MPa) and the least strength (8.2±4.1 MPa). These results correspond with those of previous work in long bones, in that periosteum interfacing with ligament or muscle (e.g. zygomatic, metacarpal) demonstrates greater stiffness and strength than periosteum adjacent to loose connective tissue (e.g. mandibular body). Therefore, the degree to which the periosteal tissue serves as a functional interface between bone and muscle is reflected in the different failure properties of periostea from different bones. The structural fortification of the zygomatic arch periosteum relative to other periosteal tissues suggests a role for the periosteum in stabilizing the zygomatic arch-muscle functional complex. On the other hand, the similar failure properties of zygomatic and squamosal periostea from the zygomatic arch mean that the differential growth of these bones cannot be attributed to mechanical stimuli intrinsic to the periosteal tissue.

Differential response of condylar, epiphyseal, synchondrotic, and articular cartilages of the rat to varying levels of vitamin A: Its impact on current growth concepts

A dietary experiment, in which the effects of varying levels of vitamin A on fifty-one Wistar rats were studied, has given new corroborating evidence that condylar cartilage responds to the experimental conditions like an active skeletal growth center and not like articular cartilage. The synopsis of the effects of hypovitaminosis A and hypervitaminosis A on the processes involved in the endochondral ossification of the condylar, epiphyseal, synchondrotic, and articular growth cartilages (Table III) revealed the unique position of the condylar cartilage inferred earlier from endocrine and orthopedic experiments. 5 It responded to the experimental conditions with effects opposite those in the epiphyses. Articular cartilage was found to respond more like an epiphyseal cartilage, but to a lesser extent. The mode of action of vitamin A on skeletal morphogenesis pointed to a possible trifold mechanism: (1) a direct interaction with the osteoblastic tissue, (2) a secondary hormonal mediation due to a primary influence on adrenal, anterior pituitary, and thyroid functions, and (3) a direct influence on the sulfate and hexosamine metabolism involved in the synthesis of amino sugars of ground substances. The hormonal mechanism was shown to be best qualified to explain the differential response of the condylar cartilage. The impact of these findings on present-day orthodontic thinking emphasizes the precedence of the inductive experimental approaches in modern biology before any deductive postulation such as that implied in the “functional matrix” concept.

Oral effects of injected strontium 90.

Studies were carried out on the jaws of rabbits injected with Sr/sup 90/ Cl/sub 2/ in single doses, varying between 100 and 1000 mu c/kg, in weanlings and animals. The rabbits were ktlled an hour to 9 months after injection; histological studies were done on one mandible and dosimetric studies were performed on the opposite mandible. The most severe changes in teeth and surrounding tissues were produced weanling rabbits receiving high injected doses (>600 mu c/kg). In this group, pyknotic cells were seen among differentiating odontoblasts after 24 hr (accumulated dose 150 rad) and pulp cells became separated from the internal enamel epithelium by an exadate. After 14 days (2500 rad) depletion of pulp cells in this area was evident. At 30 days growth of the cheek-teeth had almost stopped (4500 rad) and there was fibrosis of the pulps. The recently formed parts of the teeth were of diminished diameter. The younger odontoblasts showed great disorder and the mature parts of the pulps becarne completely replaced by dentine. Production of enamel and growth of the enamel organ were arrested, but the enamel organs did not completely disappear. No neoplastic change was seen in any of the dental tissues with themore » possible exception of cementum. In rabbits injected as adults with high doses, effects occurred which were in general similar to those seen in the weanlings, but took place after a longer time or were less severe and lasting. In those inlected at under 2 yr, growth and eruption of teeth were generally arrested or much retarded, as in animals injected as weanlings; but in rabbits injected at over 3 yr, continued growth and eruption of teeth were not so much retarded; so that in these, by 6 months after injection, most or all of the dentine which had contained the heaviest burden of Sr/sup 90/ had been lost by attrition. The quality of the tooth pulps and dentine in these older rabbits also showed a high degree of recovery from damage. Pleomorphic proliferation of osteoblastic tissue was seen much later in rabbits injected as adults than in those injected as weanlings-----at 6 months instead of at 2 or 3 months after injection. Dosimetry results showed that this was not due to decreased dose rates in the area concerned, for these and the calculated accumulated doses were much increased. Thus, in the weanling group, the max accumulated dose between the bases of the cheek teeth at the time that osteoblastic proliferation was evident was of the order of 9000 rad but in the adult group 47,000 rad or more. Whereas osteosarcomas were evident in four out of four rabbits of the weanlings series at 6 months after injection, they could not be recognized in the adult group at 6 months and only in one rabbit 9 months after injection. In this animal injected at 21 months of age the max accumulated dose between the bases of the mandibular cheek-teeth was approximates 123,000 rads, compared with the 20,000 to 50,000 rads associated with osteosarcoma of the mandible in the weanlings. The results show that, although there are in the rabbit injected as an adult preferential locations of Sr/sup 90/ in the jaws and especially the teeth compared with the distribution in weanlings, and much higher dose rates in the jaws in all areas initially and in many areas throughout, the development of sarcoma in the jaws is slower. It is not known whether this is due to different reactivity of older tissues, higher dose rate, or other factors. (BBB)« less