Change in bone mass after Colles' fracture: a case report on unique data collection and long-term implications.

The cast immobilization of a fractured limb results in a loss of bone mass; however, the long-term implications of that effect with regard to bone mineral status, particularly in other skeletal sites, are less known. The purpose of this study was to describe changes in bone mass in different skeletal sites triggered by Colles' fracture. The case is unique regarding the existence of baseline measurements taken just a few days before the fracture on all measurable skeletal sites, including the fractured radius. Therefore, it was also possible to determine whether the injury caused long-term bone loss in the affected and unaffected skeletal sites. The patient was a healthy, premenopausal Caucasian woman, in her late forties, who fractured her nondominant wrist as a result of low-impact fall on ice. The arm and the metacarpals were immobilized to the elbow for 5 wk. Bone mass measurements were performed with DPX-MD densitometer (Lunar Corp. Madison, WI) at baseline and 5, 10, 13, 21, and 52 wk postinjury. At the 5-wk measurement (on plaster removal) there was a notable increase in bone mineral density (BMD) and bone mineral content (BMC) in all sites of ulna and radius of the injured forearm (from 10 to 73%), followed by the apparent decline to or below the baseline at 10, 13, 21 and 52 wk of follow-up. Other skeletal sites were measured at 10 wk when a substantial decrease in BMD and BMC in some of the hip regions and lumbar spine was noticed; most notably in L3-L4, Ward's triangle, and femoral neck (from 2 to 8%) and remained such after 1 yr. Although this patient had a normal bone mineral status and no osteopenia detected before fracture, the trauma of radial fracture caused long-standing bone loss in fracture-prone areas-hip and spine. Because about 70% of bone strength is explained by its mineral density, the patient might be at increased risk for fracture later in life. The changes in bone mass after injury should be monitored and interpreted carefully, and more elaborate treatment of patients presenting with wrist fractures are needed to prevent any potential risk for later osteoporotic fractures in spine and hip and possible refracture of the injured extremity.

Adenovirus-mediated gene transfer into tendon and tendon sheath.

In this study, we successfully transferred the Escherichia coli beta-galactosidase gene. LacZ, into the chicken tendon and tendon sheath by a recombinant adenovirus. The recombinant adenovirus Adv-beta gal that carried the E. coli LacZ gene was constructed by homologous recombination in 293 cells (human transformed embryonic kidney) between the expressing vector and the ClaI large fragment of adenovirus 5 genome. Each chicken received a 10 microliters injection containing 10(5) plaque-forming units of recombinant virus Adv-beta gal. into the tendon sheath of the long toe Samples of tendon and tendon sheath were harvested at 3.30, and 75 days after the injection. The LacZ gene transfer was detected for its coding product beta-galactosidase by staining with X-gal solution. The results showed that all tendon and tendon sheath samples from the three harvest times stained positive (blue). The tendon sheath samples were more extensively stained; staining of the tendon was limited to the epitenon layer. These data suggest that a functional exogenous gene can potentially be transferred into the tendon and tendon sheath by similar techniques; such techniques may be used to improve healing and reduce adhesion formation.

Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Astrocyte response and transforming growth factor-beta localization in acute spinal cord injury.

STUDY DESIGN: An experimental histologic and immunohistological investigation of acute spinal cord injury was performed in a rat model. OBJECTIVE: This study determined (1) the immediate cellular and molecular responses within the spinal cord that result from a clinically relevant compression injury, (2) the acute astrocytic response to injury using the astrocyte specific GFAP antibody, and (3) the temporal pattern of cellular and extracellular localization of transforming growth factor-beta 1 (TGF-beta 1) within the spinal cord injury zone immediately after injury. SUMMARY OF BACKGROUND DATA: Ultimate neurologic outcome from spinal cord injury results from both the primary mechanical trauma and a subsequent cascade of cellular and molecular events that are termed the secondary injury. Efforts aimed at improving neurologic outcome may depend on the manipulation of cellular and molecular mechanisms that are responsible for propagating this secondary injury cascade. Astrocytes and TGF-beta are two potentially key components of this secondary injury. METHODS: Twenty-one Sprague-Dawley adult rats underwent open thoracic spinal cord injuries using the Allen weight-drop technique. Spinal cord specimens were harvested at 0, 1, 2, 4, 8, 24, and 72 hours after injury for histologic and immunohistochemical evaluation. Harvesting of injured and surrounding uninjured cord was performed before sectioning in sagittal and transverse planes. These paraffin-embedded sections were stained with polyclonal antibodies against glial fibrillary acidic protein (GFAP, an astrocytic cytoskeleton marker) and TGF-beta 1. RESULTS: A complex astrocytic response to the spinal cord injury was found within 24 hours of injury. Both the geographic and temporal patterns of astrocyte localization suggest a role in the regulation of spinal cord injury propagation. High concentrations of extracellular TGF-beta were seen immediately after injury within the hematoma at the zone of impact. Subsequently, intracellular TGF-beta was seen in astrocytic nuclei and cytoplasm, intramedullary and extramedullary capillary endothelial cells, and in motor neurons. CONCLUSIONS: The neurologic outcome in patients with SCI results in part from a secondary injury whose cellular and molecular mechanisms are poorly understood. This study suggests that both astrocytes and TGF-beta are involved in the regulation of spinal cord secondary injury. An improved understanding of their specific roles may result in novel treatments to improve the outcome from SCI.

Genetic expression of extracellular matrix proteins correlates with histologic changes during fracture repair.

We characterized gene expression in the reparative callus that formed after fracture of the rat femur. The callus was divided into regions of bone formation (hard callus) and cartilage formation (soft callus), and gene expression was examined separately in each region. Expression of extracellular matrix protein genes varied with the progression of repair and differed between hard and soft calluses. Messenger ribonucleic acids (mRNAs) for osteonectin, alkaline phosphatase, and type I procollagen were detected in the hard callus at maximal levels during endochondral ossification and bone remodeling (day 15) and at 50% maximal levels during intramembranous bone formation (day 7). Messenger RNAs for these proteins in the soft callus were detected at low levels during chondrogenesis (day 9) but increased to 80% of maximal levels with chondrocyte hypertrophy and mineralization of the cartilage matrix (day 13). Messenger RNAs for type II procollagen and proteoglycan core protein were detected at maximal levels in the soft callus during chondrogenesis (day 9). Osteocalcin gene expression was detected in the hard callus during endochondral ossification and remodeling but not during intramembranous bone formation or at any time in the soft callus. Osteonectin mRNA was detected in both the hard and soft callus throughout the entire course of fracture repair. Expression of cartilage and bone-related genes correlated with the temporal sequence of histologic changes, suggesting transcriptional regulation of gene expression during repair. Differences in gene expression between hard and soft callus and in each of these regions as repair progressed suggest local regulation of gene expression during cell differentiation and matrix synthesis.

Evaluation of metaiodobenzylguanidine heart and lung extraction fraction by first-pass analysis in pigs.

Metaiodobenzylguanidine (MIBG) is a norepinephrine analog that can be used to study cardiac sympathetic innervation. Most of the kinetic data on MIBG, however, have been obtained in vitro from adrenal chromaffin cells. To elucidate MIBG cardiac kinetics in vivo, we measured the first-pass extraction fraction (EF) of MIBG in pig heart and lungs and determined the relationship between the cardiac EF and myocardial blood flow (MBF) before and after dipyridamole, cocaine and imipramine. The first-pass lung EF was 24% +/- 0.80% (mean +/- s.e.). The baseline cardiac EF of MIBG was 79% +/- 1.6%. With dipyridamole, MBF increase significantly and the EF fell (82% +/- 2.5% to 71% +/- 3.5% baseline compared to 0.03 mg/kg/min dipyridamole, p less than 0.001), indicating that the cardiac EF of MIBG is dependent on MBF. Cocaine infusion had no effect on MBF or EF. Imipramine caused a significant increase in the EF (72% +/- 3.5% versus 77% +/- 2.5%, baseline versus imipramine p = 0.032) without a change in MBF. In adrenal chromaffin cells, cocaine and imipramine decrease MIBG uptake, suggesting that adrenal chromaffin cells may be an inappropriate model for studying MIBG kinetics in cardiac sympathetic neurons.

Transforming growth factor-beta in calf articular cartilage organ cultures: synthesis and distribution.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to play a prominent role in controlling proteoglycan synthesis and breakdown as measured following addition to organ cultures of calf articular cartilage (Morales, T. I., and Roberts, A. B., J. Biol. Chem., 263, 12,828-12,831, 1988). In this study, we compare two closely related TGF-beta isoforms, TGF-beta 1 and TGF-beta 2, both by assessing the effects of exogenous peptide as well as by analyzing the biosynthesis and total amount of these two isoforms in cartilage explants. Added exogenously, TGF-beta 1 and TGF-beta 2 induce a comparable increase in proteoglycan synthesis over basal controls with saturation at approximately 5 ng/ml. Synthesis of TGF-beta by basal calf cartilage cultures is demonstrated by (i) immunolocalization of intracellular TGF-beta, (ii) Northern blot analysis of steady-state mRNA levels, and (iii) immunoprecipitation of metabolically labeled TGF-beta from tissue extracts and conditioned culture medium. The net amount of extractable TGF-beta 1 and TGF-beta 2 in the basal cartilage cultures was assessed by a functional assay involving inhibition of proliferation of CCL-64 mink lung epithelial cells and by sandwich enzyme-linked immunosorbent assay. The predominant isoform was TGF-beta 1 (60-85%) and the total TGF-beta 1 + TGF-beta 2 was in excess of the amount required for maximal activation of proteoglycan synthesis. The level of both isoforms was maintained relatively constant between Days 2 and 7 of culture despite a sharp (approximately two to fourfold) drop in proteoglycan synthesis. This suggests that cartilage contains a large pool of TGF-beta which is not readily accessible to the chondrocyte. We propose that much of the polypeptide is sequestered in the matrix awaiting release upon demand.

The use of polymerase chain reaction generated nucleotide sequences as probes for hybridization.

In this report a rapid, simple and economical means of preparing a cDNA probe of specified length, sequence and specific activity is described. The process involves the use of a polymerase chain reaction to incorporate radiolabelled nucleotides into a single stranded or double stranded cDNA sequence. A pair of oligonucleotide primers are synthesized, flanking the sense and antisense strands of a selected sequence. The primers are then used with a cloned DNA fragment or a cellular source of RNA or DNA as a template to amplify the specific gene sequence. The sequence to be used as a probe is selected from the known sequence using free energy calculations of the secondary structure. The calculation of free energy predicts regions of stable secondary structure which may hinder transcription and thus are to be avoided. By selecting the distance between primers the probe length can be controlled to allow adequate probe permeability into tissue samples. The specific region of the gene sequence can be chosen to differentiate between closely related sequences by avoiding areas of homology. Altering the concentration of a radiolabelled nucleotide allows direct control of probe specific activity. The use of asymmetric PCR allows the preferential generation of an antisense single stranded cDNA sequence for a higher sensitivity in the detection of low abundance mRNA. This report highlights the advantage of this technique in generating probes for in situ hybridization. However, any technique that relies on homology for detection of sequences, such as Northern and Southern blotting could also utilize this technique.

Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur.

We have investigated the ability of exogenous transforming growth factor-beta (TGF-beta) to induce osteogenesis and chondrogenesis, critical events in both bone formation and fracture healing. Daily injections of TGF-beta 1 or 2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis. After cessation of the injections, endochondral ossification occurred, resulting in replacement of cartilage with bone. Gene expression of type II collagen and immunolocalization of types I and II collagen were detected within the TGF-beta-induced cartilage and bone. Moreover, injection of TGF-beta 2 stimulated synthesis of TGF-beta 1 in chondrocytes and osteoblasts within the newly induced bone and cartilage, suggesting positive autoregulation of TGF-beta. TGF-beta 2 was more active in vivo than TGF-beta 1, stimulating formation of a mass that was on the average 375% larger at a comparable dose (p less than 0.001). With either TGF-beta isoform, the dose of the growth factor determined which type of tissue formed, so that the ratio of cartilage formation to intramembranous bone formation decreased as the dose was lowered. For TGF-beta 1, reducing the daily dose from 200 to 20 ng decreased the cartilage/intramembranous bone formation ratio from 3.57 to zero (p less than 0.001). With TGF-beta 2, the same dose change decreased the ratio from 3.71 to 0.28 (p less than 0.001). These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGF-beta to proliferate and differentiate, as occurs in embryologic bone formation and early fracture healing.

Transforming growth factor-beta in the regulation of fracture repair.

The complexity of the fracture repair process has been apparent to both clinicians and scientists since its first histologic description. How fracture repair is regulated, however, remains unclear. Transforming growth factor-beta (TGF-beta), one member of a class of proteins known as growth factors, may be involved in the formation of bone and cartilage. Our experimental findings implicate TGF-beta as an important regulator of cell proliferation, differentiation, and synthesis of extracellular matrix. Further elucidation of how TGF-beta regulates bone physiology is necessary to improve therapy in pathologic conditions.

Biomechanical and histologic evaluations of pulley reconstructions in nonhuman primates.

A2 pulleys were reconstructed in nonhuman primates using expanded polytetrafluoroethylene, woven nylon, and fascia. Biomechanical and histologic evaluations were done after death at 18 weeks. Tendons were normal after all pulley reconstructions. Polytetrafluorethylene had a greater breaking strength than woven nylon or fascia, or control pulleys, both at 18 weeks and before implantation. Histologic analyses revealed fibrous ingrowth of host tissues, no adhesions, no trauma to underlying flexor tendons, and the absence of an inflammatory response, for all pulley types.

Estrogen receptor mRNA expression in callus during fracture healing in the rat.

Estrogen has profound effects on bone metabolism, yet its precise mechanism of action remains unknown. Several recent investigations have located the estrogen receptor (ER) on osteoblast-like cells, suggesting a potential direct action of estrogen via its own receptor on bone cells. The protective role of estrogen on bone in osteoporosis is well known; however, neither the existence nor the mechanism of an estrogenic role in fracture healing has been well studied. In this investigation we used a modification of polymerase chain reaction (PCR) amplification to detect and quantify ER messenger RNA (mRNA) levels in rat fracture callus previously undetectable by northern analysis. Verification of correctly amplified cDNA fragments was provided by restriction digestion. PCR failed to detect ER mRNA in total RNA extracts from tendon and fibrocartilage. The ER message was present in fracture callus at levels up to 70% of that found in rat uterus, and demonstrated a transient 13-fold increase in transcription by day 14 with return to baseline by day 31 of fracture healing. These data demonstrate that the rat estrogen receptor gene is expressed in fracture callus and suggest that estrogen may play an important role in the normal fracture healing process. In addition, this work adds to the growing body of evidence which supports a possible direct action of estrogen via its receptor on osteogenic cells.

Quantification of the perfusion of the anterior cruciate ligament and the effects of stress and injury to supporting structures.

The perfusion of the normal ACL was quantitated using the hydrogen washout technique in a canine model. This was compared to the perfusion of the synovium in the suprapatellar pouch. Changes in the ACL perfusion were quantitated after the application of anterior stress, division of the infrapatellar fat pad, and dissection of the synovium enveloping the ACL. The ACL is relatively hypovascular, with one-half the blood flow of the synovium of the suprapatellar pouch. Application of an anterior stress diminishes the blood flow to the ACL to one-fifth of the baseline value, an effect which is reversible. Division of the infrapatellar fat pad causes a two-fold decrease in perfusion to the ACL, whereas dissection of the enveloping synovium results in a complete cessation of blood flow.