James T. Bronk

Permeability of cortical bone of canine tibiae

Movement of interstitial fluid in cortical bone is considered one of the important mechanisms affecting bone remodeling and fracture healing. To better understand such interstitial fluid movement, the hydrostatic permeability of cortical bone of the canine tibia was measured in this study. It was found that age has significant influence on permeability. The cortical bone permeability of puppy tibiae is six times higher than that of adult tibiae. Adult tibial cortex is impermeable unless the superficial layer of the periosteal cortex is removed. The permeability differs between regions and locations of the tibia. The pathways for such extravascular interstitial fluid movement were also identified to be primarily the haversian and Volkmanns canals and secondarily the canalicular and lacunar spaces. The high permeability of their cortical bone may explain the increase in periosteal new bone formation seen in puppies when a venous tourniquet is applied.

Osteoblasts Express Types I and II Activin Receptors During Early Intramembranous and Endochondral Bone Formation

Increasing evidence suggests a potential role for activin in bone formation. However, the cognate receptors through which activins function with respect to skeletal tissues have not yet been identified. Identification and regulation of expression of these receptors are necessary prerequisites to understanding the role of activins in bone metabolism. We detected mRNAs for three activin receptors, type I (ActRI), type II (ActRII), and type IIB (ActRIIB), in multiple skeletal tissues in rat, including tibia and costochondral growth plate, and also in cultured osteoblasts. To gain information about the relationship between receptor expression and different skeletal cell functions, we evaluated expression of the three receptors in a semiquantitative manner during the early stages of fracture healing, a model for rapid bone formation. Relatively high levels of ActRI and ActRII expression were detected in the callus at 7, 10, and 14 days after fracture, times that correlate with the interval of rapid intramembranous bone formation and the initiation of endochondral bone formation. Expression of the ActRIIB in the fracture callus was strikingly lower than either ActRI or ActRII. Immunostaining of the fracture callus and the newborn rat femur with an anti‐ActRII antibody localized the receptor to osteoblasts at regions of membranous and endochondral bone formation. No staining of osteoblasts in fracture callus or bone was seen with an anti‐ActRIIB antibody. These results provide strong evidence of the identification of the principal receptors through which activins could function in the skeletal system and further shed light on activins mechanism of action in bone formation.

Venous pressure and bone formation

We have studied the relationship of increased venous pressure to formation of periosteal new bone in growing dogs. The methods used were measurement of fluid spaces in bone by steady-state tracer techniques, measurement of venous pressure, and measurement of the rate of periosteal new bone formation by histomorphometry. The results of paired comparisons--test versus control tibia--show an increase in venous pressure, a decrease in vascular space, an increase in extracellular fluid space (sucrose space), and an increase in periosteal new bone formation on the side of increased venous pressure. The data support the hypothesis that an increase in venous pressure results in an increase in passage of fluid from capillary to bone matrix. Increased extravascular perfusion could be a factor in increasing periosteal bone formation. This flux of fluid may increase streaming potentials in bone and these act as a signal to bone cells to increase bone formation.

Effect of weight-bearing on healing of cortical defects in the canine Tibia

UNLABELLED It has been generally accepted that mechanical stimulation is an important factor in the promotion of formation of bone. Fracture-healing consists of periosteal bridging of the fracture, which achieves stability, and proliferation of endosteal bone to fill the defects between the ends of the bone. To evaluate the effect of weight-bearing on bone-healing, an operatively created defect in the tibial cortex was chosen as an experimental model. In one set of dogs (Group 1), a bilateral defect in the tibial cortex was created and weight-bearing was permitted on one tibia but not on the opposite one. In Group 2, a bilateral defect in the tibial cortex was made and weight-bearing was allowed on both tibiae. A third group of dogs of similar age (Group 3) had no tibial defects. Quantitative histomorphometry was used to measure formation and porosity of bone. Weight-bearing was measured with both static and dynamic techniques. Significantly less woven bone formed in the defects in the non-weight-bearing tibiae than in the weight-bearing tibiae. This appeared to be due to a disuse response in the underloaded tibiae, in which less bone formed, rather than to the formation of more bone in the weight-bearing tibiae. The data suggest that weight-bearing is a permissive factor, not a stimulus, for formation of woven bone in a tibial defect. CLINICAL RELEVANCE This animal model supports the concept that lack of weight-bearing decreases the amount of woven bone that is formed in a healing tibial defect. The results of this study indicate that weight-bearing increases the formation of bone in fracture-healing.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous parathyroid hormone induces cortical porosity in the rat: effects on bone turnover and mechanical properties.

We examined the time course effects of continuous PTH on cortical bone and mechanical properties. PTH increased cortical bone turnover and induced intracortical porosity with no deleterious effect on bone strength. Withdrawal of PTH increased maximum torque to failure and stiffness with no change in energy absorbed.

Osteoblastic and Osteolytic Human Osteosarcomas can be Studied with a new Xenograft Mouse Model Producing Spontaneous Metastases

There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.

Identification of estrogen-regulated genes during fracture healing, using DNA microarray.

Estrogen deficiency impairs fracture healing, while estrogen treatment of ovariectomized rats accelerates fracture healing. To identify genes regulated by estrogen during fracture healing, we evaluated gene expression in calluses from three groups of rats: sham-operated, ovariectomized, and ovariectomized and treated with estrogen. RNA from calluses harvested 10 days after fracture was subjected to DNA microarray-based analysis of 5147 genes. In total, 52 genes were identified whose mRNA expressions were found to be downregulated by ovariectomy but restored with estrogen. Differential mRNA expression of 4 genes (collagen type 2, extracellular superoxide dismutase, urokinase-type plasminogen activator [u-PA], and ptk-3) was confirmed by reverse transcription polymerase chain reaction. Further, chondrocytes and chondroclasts were positive for u-PA in the junction between cartilage and bone, implying its importance in resorption and remodeling of callus. Identification of these genes is a prerequisite to understanding the mechanism by which estrogen influences the complex metabolic process of fracture repair.

Reaction of the circulatory system to injury and regeneration.

The anatomy of the bone blood supply is well known. Techniques to measure bone blood flow are available, but no accurate method is available for clinical use. Ion movement from the capillary to the matrix is by diffusion, and efflux appears to be controlled by a concentration-dependent binding mechanism in the extravascular space of bone. The resistance vessels of bone are regulated by local, neural, and humoral factors. Fractures of long bone provoke a decrease in blood flow followed by a large increase in blood flow driven by metabolic demand. Fractures heal by coordinated formation of new bone, which is a combination of interstitial and surface remodeling. Age affects bone remodeling. Fixation of an experimental fracture may modify blood flow as well as bone remodeling. Fluid fluxes in bone may produce streaming potentials that in turn stimulate the osteoblasts to form new bone.

Pregnancy associated plasma protein-A is necessary for expeditious fracture healing in mice

Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that regulates IGF bioavailability in vitro through cleavage of inhibitory IGF-binding protein-4 (IGFBP-4), has been implicated in skeletal development and injury repair responses. However, direct in vivo data are lacking. In this study, we used PAPP-A knock-out (KO) mice to determine the role of PAPP-A in fracture repair. Stabilized mid-shaft fractures were produced in femurs of 3-month-old mice. At 14 days post-fracture, complete bony bridging of the fracture callus was seen radiographically in wild-type but not in PAPP-A KO mice. Histological examination 5 to 28 days post-fracture showed reductions in the amount of intramembranous bone formation, cartilage production, endochondral ossification and remodeling in PAPP-A KO compared with wild-type mice. However, fracture healing appeared similar in both groups at 42 days post-fracture when analyzed by histology. A similar degree of healing strength in wild-type and PAPP-A KO femurs was demonstrated by mechanical testing at 28 and 42 days post-fracture. Untreated cultures of day 5 fracture calluses from wild-type mice showed robust IGFBP-4 protease activity and IGF receptor phosphorylation, whereas fracture calluses from PAPP-A KO mice had no IGFBP-4 protease activity and reduced IGF receptor phosphorylation. These data demonstrate a marked delay in fracture healing in PAPP-A KO compared with wild-type mice, and suggest that PAPP-A is necessary in the early phases of the process for expeditious fracture repair. The ability of PAPP-A to enhance local IGF action may be an important mechanism for optimizing the fracture repair response.

Canine Bone Blood Flow Measurements Using Serial Microsphere Injections

The objective of this study was to determine the reproducibility of serial bone blood flow (BBF) quantitation using multiple microsphere injections. Three consecutive estimates of BBF were obtained, using 15-mu radionuclide-labeled microspheres from 21 anesthetized adult dogs. A dose of 3 million spheres/kg was used in seven dogs (Cohort 1); a dose of 0.5 million spheres/kg was used in the remaining 14 dogs (Cohorts 2 and 3). Estimates of BBF were made at an average of 129, 153, and 175 minutes after the animals had been anesthetized in the first two cohorts and 179, 203, and 225 minutes in Cohort 3. The dogs in Cohort 1 had no surgical intervention; the dogs in Cohorts 2 and 3 had increasingly complex surgical interventions. Despite stabilization of cardiovascular status, BBF was found to vary by 33.4% in Cohort 1, 25.7% in Cohort 2, and 42.5% in Cohort 3 over the three injections. Cortical BBF fell by 13.9%, 12.1%, and 12.4% between the first and second, and by 31.0%, 11.2%, and 29.9% between the second and third estimates for Cohorts 1, 2, and 3, respectively. Variation in right to left blood flow was used as an overall measure of error caused by technique and did not consistently increase between the first, second, or third BBF estimates in any cohort. Cortical BBF data were found to be significantly more reliable than cancellous data (p < 0.01); error caused by technique was least in the midshaft femoral or midshaft humeral cortical samples. Increasing the dose of spheres administered from 0.5 to 3 million/kg for three serial microsphere injections increased the number of reliable samples and did not lead to increased technical error or shunting. This study demonstrates that there is a significant decrease in BBF over time in the anesthetized dog; therefore, serial estimates of BBF can only be interpreted if the results are normalized or if a control group of animals is included.