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A cylinder of gelatin containing silver spongy granules was placed in and lay within the masseter muscle, the periosteum, and the mandible, and terminated in the medial pterygoid muscle of young (3 month-old) growing miniature pigs. On the basis of an animal sacrificed one week after placement of the cylinder, it was found that the suspending gelatin was removed by cellular activity. Nine months later the remaining animals were sacrificed. Periodic X-rays were taken during the course of the experiment. After sacrifice, the mandible and associated tissues were histologically examined. The results of this study suggest that the silver granules in the muscles maintained their location during growth; the silver granules in the mandible moved forward with mandibular growth. "Slippage" appeared to occur external to the fibrous layer of the periosteum; the site of movement was revealed by the trail of the silver granules. The described method should prove of value in studying the growth interrelationships between bone, periosteum, and muscle.
Anat Rec 1979 Aug
PMID:"Silver dust"--a tool to study growth interrelationships between bone, periosteum and muscle. 47 15

Repair of a fractured membrane bone, the quadratojugal (QJ), has been studied in the newly hatched chick. Complete open fractures never united by bony fusion, even in birds maintained for six months post-fracture. Extraperiosteal connective tissue invaded the fracture gap and formed thick fibrous bundles which stabilised the fracture. Cartilage of two types formed on these bones. One was derived from periosteal cells and the other from osteoblasts or osteocytes. Considerably more cartilage formed in bones partially fractured than in those completely fractured. The "periosteal" cartilage did not form if the periosteum was removed at the time the bone was fractured. This was because, although the fibrous layer of the periosteum regenerated, the cambial layer did not. Metaplastic cartilage did form in the absence of the periosteum. Isolating fractured bones within polyethlene or glass tubes prevented accumulation of a blastema between the bony fragments. Cartilage did not form inside the tubes but did form where the ends of the tubes abutted onto the bones. Large defects in the bones (4 mm gaps, 4 mm of bone in the place of the QJ) healed via fibrous union with minimal osteogenesis and no chondrogenesis. Severing M. depressor mandibulae at the time the bone was fractured inhibited chondrogenesis, favoured osteogenesis and resulted in development of a pseudarthrosis. The potential for differentiation of the cells of the QJ and the role of the adjacent tissues as they related to repair of the fracture was discussed, and the ability of cells from membrane bones to become chondrogenic emphasized.
Anat Rec 1975 Jan
PMID:The repair of fractured membrane bones in the newly hatched chick. 110 63

The sexual dimorphism of the innominate bone was examined in 14 strains of mice. In female mice of all strains, the pubis was significantly longer and thinner than that in the strain-matched males. In 13 of 14 strains, the ischium in the male was longer and thicker than in the female. In the testicular-feminized male (Tfm) mouse, the ischium was longer and thinner than that in the wild-type male, resembling that of the wild-type female. The pubis of the Tfm mouse was longer than in the wild-type males. The pubis width in the Tfm mouse was between those of the wild-type male and female. Gonadectomy at ages of 5, 10, 20, 30, and 60 days in both sexes showed that the ischium develops as the female type when sex hormones are absent. In contrast, postnatal testicular androgen induces the male-type ischium. Gonadectomy at 60 days had a slight effect on the pubis, indicating that sexual dimorphism of the pubis was determined before 60 days of age. Estrogen receptors (ER) were immunohistochemically demonstrated in bone cells of 0- to 60-day-old mice. ER was found exclusively in the periosteum of the pubis at the day of birth; however, it appeared in bone cells of all parts of pelvis at 10-60 days. These results indicate that sexual dimorphism of the pubis is consistent for the 14 mouse strains examined, and that the shape of the pubis is determined by sex steroids before 60 days of age. Since ER exist in the bone cells, morphogenesis of the pelvis may be regulated by these sex steroids.
Anat Rec 1992 Dec
PMID:Effects of sex steroids on the development of sexual dimorphism in mouse innominate bone. 145 56

Tenascin is a glycoprotein of the extracellular matrix, which has been associated with differentiation of hard tissue forming cells. Alkaline phosphatase (AP) is involved in calcification, and it has also been suggested to function in cell differentiation. We have compared the distributions of tenascin and AP in the developing skull and teeth of embryonic and growing rats and mice. Tenascin was localized by immuno-Peroxidase and AP by enzyme histochemical staining of tissue sections. Both tenascin and AP were largely restricted to bone, cartilage, and teeth. In cartilage, tenascin was expressed in the perichondrium, whereas AP activity was detected only in the hypertrophic cartilage. In growing intramembranous bone, tenascin and AP were expressed in the periosteum and endosteum. AP activity was restricted to the inner layer of the periosteum, whereas tenascin expression extended to the more superficial layers. In bud-staged teeth tenascin but no AP activity was localized in the condensing mesenchymal cells around the epithelial bud. At the bell stage both tenascin and AP activity were localized in the cuspal mesenchyme, and the intensity of staining decreased towards the cervical region. In summary, tenascin was present at all sites of AP activity except in the epithelial cells of the enamel organ and the hypertrophic cartilage of the mandibular condyle. In mesenchymal tissues tenascin was more widely distributed than AP. It can be suggested that tenascin has functions at earlier stages of hard tissue formation than AP.
Anat Rec 1990 Sep
PMID:Comparison of the distribution patterns of tenascin and alkaline phosphatase in developing teeth, cartilage, and bone of rats and mice. 170 Jun 48

The present study identifies, localizes, and reports the relative composition of specific glycosaminoglycans within tissue matrices during the initiation phase of limb regeneration. The regenerate tissues were harvested and assayed morphologically, histochemically, and chemically. We observed 1) a population of cells interspersed among the cells of the dermis, epimysium, perimysium, perichondrium, and periosteum. 2) This population was distinguishable by a unique pattern of glycoconjugate staining, i.e., intracellular and pericellular heparan sulfate and glycoproteins and extracellularly associated hyaluronate and glycoproteins. 3) Cells with these staining characteristics aggregated to a position directly beneath the apical epidermal cap. 4) Extracellular hyaluronate and glycoproteins colocalized with undifferentiated tissues. And 5) extracellular chondroitin sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans colocalized with differentiated tissues. The correlations of distinct glycoconjugate compositions with specific regeneration morphologies suggest the possibility that these components may be related to the phenotypic expression of tissues during regeneration.
Anat Rec 1989 Feb
PMID:Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma. 249 26

Intrinsic differences in bone formation rate, cell numbers, and the percentages of cells expressing alkaline phosphatase activity were studied in explants of chick calvaria periosteum cultured for 4 days and 6 days. Proliferation, differentiation, and bone production were examined in radioautographs of plastic sections and by using whole-culture biochemical assays of protein and alkaline phosphatase. Ectocranial explants at both 4 days and 6 days exhibited more alkaline phosphatase-positive cells and significantly more bone formation than endocranial cultures. There were no detectable differences in cell numbers or 3H-thymidine labeling indices. The volume of bone synthesized per osteoblast was significantly higher in the ectocranial group. Examination of bone stripped of periostea and then cultured for 4 days revealed that large areas of bone were covered by osteoblasts, indicating that the periosteal explant cultures were composed almost exclusively of osteoprogenitor cells and fibroblasts. The data suggest that the level of expression of predetermined osteogenic phenotypes can be maintained in vitro for 6 days following explantation and that variations in the rate of osteogenesis are programmed into progenitor cells prior to their differentiation into osteoblasts.
Anat Rec 1989 Jan
PMID:Site-specific regulation of osteogenesis: maintenance of discrete levels of phenotypic expression in vitro. 291 54

Osteoclast progenitors are seeded via the blood stream in the mesenchyme surrounding embryonic long bone models long before the appearance of multinucleated osteoclasts. The proliferation and differentiation of these progenitors in embryonic mouse metatarsal bones was studied with acid phosphatase (AcP) histochemistry and 3H-thymidine autoradiography. In vivo, tartrate-resistant, acid phosphatase-positive, mononuclear cells appear in the periosteum (AcPP-P cells) at the age of 17 days (after conception). On day 18, AcP-positive, multinucleated osteoclasts invade the bone rudiment and start resorbing the calcified cartilage matrix, resulting in the formation of the marrow cavity. The kinetics of osteoclast formation in vitro was studied in metatarsal bones of embryonic mice of different ages cultured in the continuous presence of 3H-thymidine. In young bones (15 days), mainly proliferating, 3H-thymidine-incorporating progenitors gave rise to AcPP-P cell and osteoclast formation. In older bones (16 and 17 days) osteoclasts were progressively more derived from postmitotic, unlabeled precursors. Irradiation of the metatarsal bones with a radiation dose of 5.0 Gy prior to culture resulted in a selective elimination of the proliferating progenitors, whereas the contribution of postmitotic precursors in AcPP-P cell and osteoclast formation remained unchanged. The results demonstrate that in the periosteum of embryonic metatarsal bones a shift occurs from a population composed of proliferating osteoclast progenitors (15 days) to a population composed of postmitotic precursors (17 days) before multinucleated osteoclasts are formed (18 days). Obviously, postmitotic AcP-negative precursors, already present in 16-day-old bones, differentiate into precursors characterized by tartrate-resistant AcP activity, the preosteoclasts (17 days), which in their turn fuse into osteoclasts.
Anat Rec 1986 Apr
PMID:Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro. 370 84

Bone cells obtained by digestion of fetal mouse or chicken calvaria were tested for their ability to form or resorb bone in vitro. The isolated cells were precultured for 6 days and subsequently cocultured for 11 days with periosteum-free noninvaded fetal mouse long bone rudiments. Bone formation and resorption during coculture were evaluated by histology and 45Ca release from prelabeled bones. The calvarial origin of cells in cocultures was traced by labeling the cells with 3H-thymidine before coculture, followed by autoradiography. Many osteoblasts and osteoclasts as well as fibroblasts developed from mouse periosteal cells released late in the sequential digestion procedure and previously denoted as "osteoblastlike" (BL). No or few osteoblasts and osteoclasts but many fibroblasts developed from early released cell fractions that have previously been denoted as "osteoclastlike" (CL). Only osteoblasts and fibroblasts but not osteoclasts developed from chicken calvarial cell fractions. The osteoblasts developed primarily from cell fractions from the inner layer of the periosteum, previously denoted as "osteoblastlike" (OB). Cells obtained from the outer layer of the periosteum (PF) gave rise mainly to fibroblasts. These studies show that osteoblast and osteoclast precursor cells are maintained in monolayer cultures of periosteal cell fractions. However, sequential digestion of mouse calvaria does not lead to separation of the two types of bone cells. Rather, osteoclast and osteoblast precursors are released jointly, from the periosteal cell layers closest to the bone surface. In the chicken cell fractions osteoclast precursors are absent after preculture, resulting in a more homogeneous population of osteoblast and fibroblast but not osteoclast precursors.
Anat Rec 1986 Jan
PMID:Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells. 395 57

Left forelimbs of postmetamorphic Xenopus laevis froglets were repeatedly denervated prior to and following amputation. Amputations were performed 14, 21, 28, or 42 days after the original denervation. A tissue-regenerative response resulting in the formation of a spike-shaped, heteromorphic outgrowth was found in the sham-denervated and control animals, but dedifferentiation of the stump tissues was not apparent. Tissue-regenerative outgrowths were not observed in the denervated cases; instead, dermal wound healing and stump and scar formation occurred. In both control and experimental cases, however, a periosteal proliferative response to amputation injury led to the development of a greatly thickened periosteum the length of the amputated radius-ulna as well as a cap of cartilage at the distal end of these bones. We conclude from these results that forelimbs of postmetamorphic froglets are incapable of adjusting to a prolonged nerveless state sufficient to allow the normal tissue-regenerative response of spike outgrowth formation.
Anat Rec 1986 Mar
PMID:Effects of denervation and delayed amputation on forelimb regeneration in Xenopus laevis froglets. 396 24

Postamputational healing was compared in nonregenerating and regenerating animals to determine whether bone healing might interfere with a regenerative response in mice. More than 150 mouse toes and 100 newt limbs were examined at the light microscope level. Stages of normal bone healing with approximate times of occurrence were established. Major differences in healing of these two species were seen. The periosteum produced hyaline cartilage, woven bone, and chondroid bone in mice, but only hyaline cartilage in newts. The endosteum produced woven bone in mice but no new growth in newts. Dead bone persisted in mice but was removed in newts. The marrow cavity became sealed in mice but remained open in newts. Despite these differences both animals produced skeletal tissue distal to the amputation plane. Woven bone formed distal to the amputation plane of mice. Cartilage formed distal to the amputation plane of newts, but cartilage was never seen distal to the plane of mice. Results of previous studies reveal that cartilage can be formed distal to the amputation plane of experimentally treated mice. Thus, although it does not regenerate, mouse bone is capable of producing, distal to the amputation plane, the type of skeletal tissue which appears at that location during an epimorphic regenerative response. This observation, in combination with other experimental results, indicates that both skeletal and soft tissues at the amputation site of treated mammals can resemble comparable tissues of newt limbs at an early stage of regeneration.
Anat Rec 1985 Feb
PMID:Bone healing after amputation of mouse digits and newt limbs: implications for induced regeneration in mammals. 397 84


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