David J. Simmons

Fracture healing perspectives.

This paper reviews the sequence of histomorphologic changes that occur in and around a fracture site, and discusses recent concepts about the roles of the cells and bone matrical moieties in promoting specific cell transformations during the phases of callus tissue formation and consolidation of the bony cortex. Current knowledge about the roles of autocrine, paracrine, and endocrine polypeptide growth and transforming factors lends new perspectives about this classic problem in bone physiology.

Formation of bone by isolated, cultured osteoblasts in millipore diffusion chambers.

SummaryOsteoblast-like and osteoclast-like cells freed from neonatal calvaria by sequential enzymatic digestion after 6–7 days in culture were placed in diffusion chambers and implanted in the peritoneal cavities of CD-1 mice. About half of the chambers also contained a dead calvarium to test for the need of an “inducer.” After 20 days, 11 of 18 chambers containing the osteoblast-like cells formed large foci of mineralized bone that corresponded to alkaline phosphatase activity throughout the chambers. Moreover, only type I (i.e., bone) collagen was formed. Occasional deposits of bone were found in only 3 of 22 chambers containing the osteoclast-like cells. The presence of dead bone did not affect any of the results. These data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.

Measurement of regional bone and bone marrow blood flow in the rabbit using the hydrogen washout technique.

This study tested the feasibility of using the hydrogen washout technique for measuring regional bone and bone marrow blood flow. Washout curves in cancellous and cortical bone were mono-exponential, indicating homogeneous perfusion of these tissues. Epiphyseal cancellous bone blood flow (0.129 +/- 0.015 ml/min/ml) and metaphyseal cancellous bone blood flow (0.170 +/- 0.014 ml/min/ml) were approximately twice as rapid as that of cortical bone (0.069 +/- 0.002 ml/min/ml). Washout of hydrogen from bone marrow was variable and usually formed a bi-exponential pattern, indicating non-homogeneous perfusion. Blood flow rate determined by analyzing the rapid components was 1.04 +/- 0.10 ml/min/ml, and that from the slow components was 0.27 +/- 0.02 ml/min/ml. Our blood flow rates are within the range of values reported by investigators using different methods, and the hydrogen washout technique offers specificity and ease of repetitive determinations not available with other methods.

Bone resorption in vitro and in vivo in PGE-treated mice.

Summary The bone-resorbing activity of prostaglandin was assessed in neonatal mouse calvaria maintained in culture as well as in the bones of adult mice in vivo. In an in vitro 45Ca-release bioassay, PGE2, the most active compound, produced a maximal stimulation of radiocalcium bone depletion of 89% versus 38% for PGF2α and 12% for PGA1. Daily intraperitoneal injection of 10 μg of 16,16-dimethyl-PGE2-methyl ester, a long-acting synthetic analog of PGE2, in several groups of C57/B1/6J mice produced a mean 33% loss of trabecular bone and a 140% increase in the area of bone lined by osteoclasts. Moreover, treatment of 4-month-old mice with di-M-PGE2 for 4 days caused a mean 15% depletion of bone calcium. There were no significant changes in blood calcium with the protocol employed.

Rhythmic dentinogenesis in the rabbit incisor: Circadian, ultradian, and infradian periods

SummaryWe have identified a variety of biological rhythms involved in the apposition and mineralization of dentin in the rabbit incisor.Animals were injected during the day or night with lead acetate at 2-week intervals—to provide biological time markers in forming dentin—and transverse undecalcified sections of the lower incisors were prepared for electron microprobe analysis. The positions of the lead markers were identified, and the continuous distribution of calcium and sulfur was measured at 1 µm intervals between the markers. In thin sections stained with hematoxylin after decalcification, the widths of a series of structural increments (bands) were measured with an ocular micrometer.Fourier analysis of the data revealed spectra of structural and compositional rhythms with a range of periodicities which extended from a matter of hours [ultradian (<24 h)] to days [infradian (>24 h) and circadian (approximately 24 h)].The structural and compositional rhythms appeared to be independent to the extent that they did not necessarily have the same periods, or amplitudes. Nor were there simple phase relationships between all of the rhythms. At some times, Ca and S fluctuations are inversely proportional (180° out of phase), but in other cases they are directly proportional or out of phase by varying degrees other than 180°. The analyses thus suggest that calcium and sulfur deposition (representing mineral and glycosaminoglycan deposition, respectively) are not simply inversely proportional, and that the hematoxylin-stained structural increments did not solely reflect differences in the distribution of the mineral components in dentin.

The effect of varus stress on the moving rabbit knee joint.

Unicompartmental osteoarthritis was produced by applying varus stress to moving rabbit knee joints. Degenerative changes were confined to the medial tibial and the medial femoral articular surfaces. Within the range of varus stress used, duration appears to be more important than magnitude of varus stress in determining the severity of cartilage damage. The calcified zone remained histologically unchanged despite advanced changes in the noncalcified zone superficial to the tidemark. Intrachondral degenerative cysts were frequently found in the basilar layers of the noncalcified cartilage adjacent to the tidemark where shear stresses were likely to be highest and diffusible nutrients least available. Highly cellular cartilaginous tissue was noted in the subchondral marrow spaces in the specimens with advanced cartilage degeneration. These areas appeared to be continuous with the overlying degenerated cartilage through gaps in the calcified cartilage. Subchondral bone did not show remarkable trabecular thickening despite advanced degenerative changes in the articular cartilage.

Osteoclast precursor cells are present in the blood of preossification chick embryos

Abstract In an attempt to ascertain the time of appearance of circulating osteoclast precursor cells, we have transplanted quail bone rudiments into or onto the extraembryonic membranes of variously aged chick embryos. We observed that osteoclast precursor cells (1) are present in the embryonic circulation prior to the onset of osteogenesis, (2) differentiate precociously in response to a factor or factors present in developing bone rudiments, and (3) increase in number until about midway in embryonic life.

The changes in quality of mandibular bone mineral in otherwise totally immobilized Rhesus monkeys

SummaryThe status of bone mineral and osteocalcin in the young adult Rhesus monkey mandible was assessed following a 14-day period of postcranial immobilization, and after 7- and 28-day recovery periods. Specimens of cortical bone taken from the compact bone at the inferior border of the jaws were ground in liquid nitrogen and sieved to a particular size below 20 μm. The bone powder was then fractionated in a bromoform-toluene density gradient to determine its mineralization profile (Ca, P, CO3, and osteocalcin), and X-ray diffraction was used to determine apatite crystal size in some fractions. There was no change in the chemistry of the mandibular bone from the immobilized animals. However, the mineralization profile in that group showed a significant shift toward the higher density fractions, indicating the presence of a greater than normal content of mature well-mineralized bone. While this trend was accentuated in the jaws following a 7-day postimmobilization recovery period, partial recovery of the normal profile was observed after a 28-day recovery period. The osteocalcin profile shifted like the mineralization profile during the immobilization and recovery periods. X-ray diffraction analyses showed that the shift in the mineralization profile during the immobilization period was associated with a decrease in apatite crystal size.

The healing of grafts combining freeze-dried and demineralized allogeneic bone in rabbits

The adjunctive role of demineralized bone matrix (DBM) in enhancing the incorporation of segmental freeze dried (FD)-allogeneic bone grafts has been studied in the rabbit ulna and fibula. The studies compared the healing patterns of fresh segmental autografts, FD-allografts, DBM-allografts, and FD-allografts supplemented at the graft-host junctions with FD- or DBM-allografts as particulates (ulna) or as segmental struts (fibula). The outcome was evaluated at five and ten weeks by a radiologic score, by biomechanical properties (breaking strength, energy to failure, stiffness), and histology. The ulnar autografts healed most rapidly (ten weeks = 100%), followed by DBM allografts (60%). By all criteria, FD-allografts were poorly incorporated (20-40%), and the process was not improved by supplements of FD- or DBM-particulate/strut bone. Histologically, the DBM component of composite fibular strut grafts was osteoinductive and united with host tissues within 35 days. The contiguous FD-allograft struts were not incorporated, showing fibrocartilaginous nonunions and resorptive foci. While the addition of DBM does not protect FD-allograft integrity in the rabbit, segmental mineralized FD-allografts could provide mechanical support for some intervals until, in such composite grafts, osteoinductive processes produced biomechanically competent new bone.

Mineralization in rat metaphyseal bone exhibits a circadian stage dependency.

Abstract Density gradient fractionation analysis of rat metaphyseal bone was used to delineate the biorhythmic changes in bone matrix mineralization. Seventy-two 4-week-old rats were entrained to 12-hr light, 12-hr dark cycles (light, 0800-2000 hr; darkness, 2000-0800 hr) for 4 weeks. All animals were fed ad lib. on Purina laboratory rat chow and tap water. Groups of 10-12 rats were killed by cervical dislocation at 4-hr intervals during a 24-hr period, and the tibias were then biopsied and frozen in liquid N2. Metaphyseal bone was fractionated via bromoformtoluene density gradients into specific gravity fractions ranging from 1.7 to 2.8. Density gradient fractions were analyzed for concentrations of calcium and inorganic phosphorus. Chronograms indicated that the accumulation of both calcium and inorganic phosphorus into the newly forming/least-dense mineral moieties of bone (1.3-1.7 sp grav) showed a single peak in the biorhythm of the rat. A statistically significant circadian rhythm of mineralization was detected for calcium (P < 0.001) and inorganic phosphorus (P < 0.039), with peaks during the environmental dark span. These results suggest that the physiological phasing of bone mineralization in the light-dark synchronized rat, is similar to that previously noted for cartilage mineralization and is antiphasal to the midday peak in bone collagen synthesis.