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Biomedical subjects

M E Holtrop

Publications and source records attributed to M E Holtrop.

At least 19 recordsLinked to original sources
PubMed1995

Long-term healing of bone using recombinant human bone morphogenetic protein 2.

A 2.5-cm-long middiaphyseal plate-stabilized segmental defect in the right femora of 5 adult sheep was implanted with 1.5 mg of recombinant human bone morphogenetic protein 2 mixed with inactivated demineralized ovine bone matrix. Bone healing was evaluated for 12 months using clinical, radiographic, gross pathologic, and histologic techniques. Bone formation within the defect was first visible radiographically between Weeks 2 and 4 after surgery; bone union was apparent between Weeks 12 and 16, at which time the plates were removed. Recanalization of the medullary cavity with neocortex formation was near completion at Week 52. Bone mineral content at the defect sites equaled that of the nonsurgically treated intact femora by Week 16. Perifemoral soft tissue mineralization did not occur, and callus size was not greater than that formed with autograft. By Week 52, the sheep were not lame, and at necropsy the surgically treated femora were rigidly healed. Woven and lamellar bone bridged the defect site. An apparently normal sequence of ossification, modeling, and remodeling events had occurred. Recombinant human bone morphogenetic protein 2 mixed with a suitable carrier could provide an alternative to autograft for use in a variety of orthopaedic procedures.

Absorptiometry, Photon↗
PubMed1993

Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein.

A middiaphyseal, 2.5-cm osteoperiosteal segmental defect stabilized by plate fixation was created in the right femur of 17 sheep. Four treatment groups were included: Group I, no implant; Group II, inactive bone matrix; Group III, recombinant human bone morphogenetic protein (rhBMP-2) mixed with inactive bone matrix; and Group IV, autogeneic bone graft. Three animals had early failure of fixation, and the remaining 14 were evaluated at three months after implantation. Radiographs showed bony union of all defects treated with rhBMP-2 (six) and a lack of bony union in the negative-control groups treated with no implant (three) and inactive bone matrix without BMP (three). Both defects treated with autograft healed. New bone formation in the defect sites treated with rhBMP-2 first appeared one month after implantation and had a mean bending strength (expressed as a percentage of the contralateral femur) of 91% +/- 59% (mean +/- standard deviation) for defects treated with BMP-2, 77% +/- 34% for autograft, 9% +/- 8% for no implant, and 11% +/- 7% for inactive matrix without BMP. Three sheep treated with rhBMP-2 had their fixation plates removed at four months and were followed for one year. Their bone defect sites remained solidly healed one year after the initial operation.

Animals↗
PubMed1991

Mandibular reconstruction with a recombinant bone-inducing factor. Functional, histologic, and biomechanical evaluation.

Bone morphogenetic protein-2 (BMP-2) is a human recombinant bone-inducing factor that stimulates bone formation within 14 days. Twenty-six dogs underwent reconstruction of 3-cm full-thickness mandibular defects. After stabilizing the defects with stainless steel reconstruction plates, test implants composed of inactive dog bone matrix carrier and human recombinant BMP-2 were placed in defects of 12 animals (group 1). Control implants (carrier without BMP-2) were used in 10 animals (group 2), and no implants were placed in mandibular defects of four animals (group 3). Animals were killed at 3 and 6 months. The reconstructed segments were evaluated by roentgenography, analysis of functional stability, histology, histomorphometry, and analysis of biomechanical strength using three-point bend testing. In group 1, reconstruction plates were removed at 10 weeks because stiff, noncompressible mineralized bone formed across the defects, allowing the animals to chew a solid diet. The defects from groups 2 and 3 showed minimal, if any, bone formation and remained grossly unstable, prohibiting plate removal or advancement to a solid diet. Histomorphometric analysis at 6 months revealed that 68% of the group 1 implants were replaced by mineralized bone, whereas mineralized bone occupied less than 4% of the implants in groups 2 and 3. Biomechanical testing at 6 months revealed that the average bending strength of the reconstructed hemimandibles (expressed as a percentage of the contralateral hemimandible) was 27% for group 1 and 0% for group 2. The biomechanical strength of the defects reconstructed with BMP-2 increased significantly from 3 to 6 months and was related to degree of mineralization and thickness of bone bridging the defect.

Animals↗
PubMed1990

The effects of fluoride on ectopic bone formation.

The effects of fluoride (F) on ectopic bone formation induced in rats by implants of demineralized cortical bone tissue were studied. Test rats received sodium fluoride (NaF), 6 mmol/liter in drinking water, and controls received fluoride-free water. Implant accumulations of tracer hydroxyproline ([3H](OH)P), 45Ca, and stable Ca were determined 24 h after injections of tritiated proline ([3H]P) and 45Ca, to estimate rates of collagen synthesis, mineralization, and net mineral mass, respectively. Conventional histology on undemineralized implant sections was done. Mineralized bone was first observed by implant histology, 2 weeks after implantation and continued to increase up to 8 weeks. A few chondrocytes were observed. Prior to bone formation, dense fibrous tissue was observed within the marrow space of the original implant. The rate of collagen synthesis peaked at 1 week, again at 3 weeks, and then continued at a slower rate up to 8 weeks. The rates of mineralization paralleled the rates of collagen synthesis between 2 and 8 weeks, indicating bone mineralization over this period. During the first 2 weeks after implantation no mineral deposition was observed. The initial peak of collagen synthesis without mineralization (0-2 weeks) indicates fibrous tissue formation and is in agreement with the histological analysis. Fluoride treatment increased rates of collagen synthesis during both the initial period of fibrous tissue formation and later bone formation. The ratio of mineralization rate to collagen synthesis rate (45Ca/[3H](OH)P) was decreased by fluoride throughout the 2-8 week period, but net mineral mass was comparable to control rats by 8 weeks, indicating that fluoride delays, but does not prevent, bone mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Effects of serum calcium and phosphorus on skeletal mineralization in vitamin D-deficient rats.

In the present study, we have evaluated the role of calcium and phosphorus concentrations in serum on the mineralization of bone in the absence of vitamin D. This was accomplished by feeding mother rats and subsequently their pups vitamin D-deficient diets varying in calcium, phosphorus, and lactose content. After 5-7 wk on these diets, serum concentrations of 25-hydroxyvitamin D [25(OH)D] and 1,25-hydroxyvitamin D [1,25(OH)2D] were undetectable. Rats fed a vitamin D-deficient diet containing 0.44% calcium and 0.3% phosphorus showed a serum calcium of 4.9-5.9 mg/dl and a serum phosphorus of 7.3-8.2 mg/dl; rickets (wide epiphysial plates) had developed as well as osteomalacia (wide osteoid seams). Rats maintained on a vitamin D-deficient diet containing 3% calcium and 0.65% phosphorus had normal serum calcium, low serum phosphorus, and severe rickets, but osteomalacia was not seen. Rats fed a diet containing 20% lactose, 4% calcium, and 1% phosphorus showed normal serum calcium, somewhat low serum phosphorus, normal serum PTH, normal width of the epiphysial plate, normal volume density of trabecular bone, and normal volume density of osteoid seams. These data confirm the findings of others, using a different experimental model, that serum calcium and phosphorus concentrations are the determining factors in mineralization defects and not the absence of 25(OH)D or 1,25(OH)2D. In these rats thyroparathyroidectomy is well tolerated, which makes for an ideal model for the study of the effects of calcium-regulating hormones on bone histology, cytology, and biochemistry.

Animals↗
PubMed1984

Effects of phosphate on calcium release, lysosomal enzyme activity in the medium, and osteoclast morphometry in cultured fetal rat bones.

The relationship between changes in medium phosphate concentration and three indices of cell-mediated resorption in fetal rat bone cultures--calcium release, the activity of the lysosomal enzyme beta-glucuronidase in the medium, and the morphology of osteoclasts--has been investigated. Bones treated with either 1 mM or 4 mM phosphate, with or without parathyroid hormone, were examined. After 2 h of culture we found the predominant effect of changes in medium phosphate to be on non-cell-mediated resorption. However, after 24 h changes in medium phosphate affected both cell-mediated and non-cell-mediated resorptive mechanisms. The 24 h effects of phosphate were not associated with either a change in the activity of beta-glucuronidase in the medium or in the area of the ruffled border of osteoclasts, but 4 mM phosphate did prevent parathyroid hormone from increasing the area of the clear zone of osteoclasts. These results imply that changes in medium phosphate alter cell-mediated resorption by affecting mechanisms that are independent of increases in beta-glucuronidase activity or changes in the ruffled border of osteoclasts but that may involve effects on the clear zone of osteoclasts.

Animals↗
PubMed1982

Cells of the mononuclear phagocytic system resorb implanted bone matrix: a histologic and ultrastructural study.

Implantation of mineral-containing bone fragments into calvarial defects in rats initiates a rapid and reproducible resorption of the bone matrix. After 7 days, a dense tissue develops with mononucleated as well as multinucleated cells surrounding and between the bone fragments. Electron microscopy revealed that these cells belong to the mononuclear phagocytic system: they were identified as macrophages, epithelioid cells, foreign body giant cells, and Langerhans cells. In addition to the common ultrastructural characteristics, these cells had electron-dense, focal specializations along their cell membrane with a coating on the exterior, corresponding to subplasmalemmal linear densities. Small, unidentified cells with electron-dense ground cytoplasm were often seen in close proximity to more differentiated cells. No halisteresis had occurred on the surfaces of the bone fragments. Indentations resembling Howship's lacunae were frequent; these contained mononucleated as well as multinucleated cells. Some surfaces were frayed and collagen fibers were exposed, but the cells apposed to these surfaces did not have ruffled borders as are seen in osteoclasts. Some bone fragments were broken up and cell processes had penetrated deep into the cracks, separating pieces of matrix. Small matrix particles were phagocytosed by macrophages, but not by epithelioid cells or giant cells. It appears that enzymes capable of degrading bone matrix components were secreted by the more differentiated cells of the mononuclear phagocytic system. They eroded the bone surface in a way reminiscent of osteoclastic bone resorption. They also entered the canaliculi to act from within the bone fragment, a process possible only in dead bone. We suggest a possible relationship of these cells with osteoclasts.

Animals↗
PubMed1981

The ultrastructure of osteoclasts in microphthalmic mice.

Osteoclasts in microphthalmic (mi/mi) mice were significantly smaller than in their heterozygous (mi/+) or normal (+/+) littermates as measured by electronmicroscopic morphometry. Clear zones were also significantly smaller and normal ruffled borders were absent. The cytoplasm was not fully differentiated. No differences were seen between osteoclasts from mi/+ and +/+ mice. The number of osteoclasts was not different in mi/mi, mi/+ or +/+ mice. Lysosomal enzymes and collagenase content of bones in the three genotypes did not show marked differences.

Animals↗
PubMed1981

1,25-dihydroxycholecalciferol stimulates osteoclasts in rat bones in the absence of parathyroid hormone.

Three experiments were carried out to test the time course of effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the ultrastructural morphometry of osteoclasts. The addition of lactose to a vitamin D-deficient diet with a high calcium and phosphate content, fed to weanling rats for 4 weeks, ensured normacalcemia and normophosphatemia and allowed thyroparathyroidectomy without ill effects. In these vitamin D-deficient thyroparathyroidectomized rats, iv injection of 50 ng 1,25-(OH)2D3 resulted in significant changes in the osteoclasts in the metaphysis of the tibiae compared to those in corresponding controls; the size of these cells, their nuclei, ruffled borders, and clear zones enlarged after 6 h and the number of osteoclasts increased after 48 h. Serum calcium and serum phosphate levels increased after 12 h in one experiment, but not in a second experiment. Serum 25-hydroxyvitamin D and serum immunoreactive parathyroid hormone levels were undetectable. Mineralization of metaphyseal bone matrix was normal, as quantified by histomorphometry. When, dependent on the mineral content in the diet, mineralization was impaired and the volume density of the osteoid seams was increased, activation of osteoclasts by 1,25(OH)2D3 was not seen until 12--24 h after injection. It is concluded that a physiological dose of 1,25-(OH)2D3 stimulates the activity of osteoclasts in the absence of parathyroid hormone.

Animals↗
PubMed1980

Human osteopetrosis: a histological, ultrastructural, and biochemical study.

UNLABELLED: Histological ultrastructural, and biochemical studies of the tissues of a patient with osteopetrosis were done. The bone contained an increased number of osteoclasts which were characterized ultrastructurally by the absence of ruffled borders and clear zones, hallmarks of actively resorbing osteoclasts. In contrast with normal human bone, tissue collagenase was not detected in osteopetrotic bone cultured in vitro, nor was tissue collagenase activity released when the osteopetrotic bone was incubated with parathyroid hormone. No striking abnormalities of parathyroid hormone or calcitonin were found in the blood or parathyroid and thyroid glands. Except for a slight increase in the extent of lysine hydroxylation of bone collagen, no significant biochemical abnormality of collagen was found. The histological, ultrastructural, and biochemical data support the hypothesis that the basic defect in osteopetrosis is cellular and the osteoclasts, in particular, are abnormal. They appear to be unable to resorb bone and cartilage, and they do not appear to respond to parathyroid hormone in a completely normal way. CLINICAL RELEVANCE: Bone marrow transplantation has produced improvement in both experimental animals and humans and should be actively considered in a child with osteopetrosis who is not thriving.

Amino Acids↗
PubMed1979

Comparison of the effects of 1,25-dihydroxycholecalciferol, prostaglandin E2, and osteoclast-activating factor with parathyroid hormone on the ultrastructure of osteoclasts in cultured long bones of fetal rats.

The effects of 1,25-dihydroxy vitamin D3 [1,25(OH)2D3], prostaglandin (PGE2), and osteoclast-activating factor (OAF) on the size of osteoclasts, nuclei, ruffled borders, and clear zones in cultured long bones of fetal rats were quantitated. In addition, the number of osteoclasts in the bones was counted and the release of calcium from the bone into the culture medium was determined. These data were compared with the corresponding effects of parathyroid hormone (PTH). All agents tested increased the size of the ruffled borders significantly after 3 h, the size of the clear zones after 12 h, and the size of the cells after 12-24 h. No important differences in sizes were noticed between the agents tested or between the agents and PTH. The number of osteoclasts was increased after 24 h of treatment with PTH, but not after the other agents. Calcium release was significantly increased for all agents between 12 and 24 h. It is concluded that bone resorption by 1,25(OH)2D3, OAF, and PGE2 is mediated primarily by increased activity of existing osteoclasts similar to PTH activation.

Animals↗
PubMed1977

Organization and cellular biology of the perichondrial ossification groove of ranvier: a morphological study in rabbits.

The perichondrial ossification groove of Ranvier, a circumferential groove in the periphery of the epiphyseal cartilage, was studied in rabbits whose ages ranged from one week to eight months using light and electron microscopy, autoradiography after labeling with 3H-thymidine, 3H-proline, and 3H-glucosamine, and histochemical staining for proteoglycans and alkaline phosphatase. By these methods, three groups of cells were identified within the groove: 1. A group of densely packed cells deep in the groove, which are the progenitor cells for the osteoblasts that form the bone bark, a cuff of bone surrounding the epiphyseal growth-plate region and the adjacent part of the metaphysis. 2. A group of more widely dispersed, relatively undifferentiated mesenchymal cells and fibroblasts, some of which are chondroblast precursors that probably contribute to appositional chondrogenesis and growth in width of the epiphyseal cartilage. 3. Fibroblasts and fibrocytes among sheets of highly oriented and organized collagen fibers which form a fibrous layer that is continuous with the outer fibrous layer of the periosteum and with the perichondrium. This layer also sends fibers into the epiphyseal cartilage and anchors the periosteum firmly to the epiphyses as bone growth proceeds.

Animals↗
PubMed1975

Actin-like filaments in bone cells of cultured mouse calvaria as demonstrated by binding to heavy meromyosin.

A variety of intracellular filaments (50-70 A in diameter) found in bone cells was shown to bind specifically to HMM. Because of this property, these filaments are probably biochemically similar to muscle actin. in osteoblasts and osteocytes, these reactive filaments were oriented in bundles parallel to the plasma membrane and filling the cell processes. In the osteoclast the filaments along the cell membrane were not so highly organized. In the clear zone, the quiescent part of the cell adjacent to the motile ruffled border, organized filament bundles were oriented perpendicular to the cell membrane and terminated in short processes at the bone surface. These filaments were also reactive with HMM. The possible significance of the filaments with respect to the physiology of bone cells is discussed.

Actins↗
PubMed1974

The effects of parathyroid hormone, colchicine, and calcitonin on the ultrastructure and the activity of osteoclasts in organ culture.

The ultrastructure of osteoclasts was examined in fetal rat bones after stimulation or inhibition of resorption in culture. A central ruffled border area completely encircled by a clear zone was considered to represent the resorbing system of the cell. The proportion of ruffled border and clear zone in osteoclast cross sections was compared with changes in bone resorption as measured by the release of previously incorporated radioactive calcium ((45)Ca). In control cultures 55% of the osteoclast cross sections showed an area closely apposed to bone and this consisted mainly of clear zone; only 11% showed ruffled borders. Treatment with parathyroid hormone (PTH) increased (45)Ca release, increased the frequency of finding areas closely apposed to bone (79%), and markedly increased the frequency of the ruffled border area (64%). Colchicine given concurrently with PTH decreased the number of osteoclasts. Colchicine or calcitonin treatment after PTH stimulation decreased the proportion of ruffled border area significantly by 1 h; this was followed by a decrease in (45)Ca release. These inhibited osteoclasts resembled osteoclasts from control, unstimulated cultures, suggesting that the cells had returned to their inactive state. Colchicine-treated osteoclasts also showed a loss of microtubules and a massive accumulation of 100 A filaments, suggesting that synthesis of microtubular subunits had increased.

Animals↗