Biomedical subjects

James T Ryaby

Publications and source records attributed to James T Ryaby.

10 recordsLinked to original sources

PubMed2007

Genetic network and pathway analysis of differentially expressed proteins during critical cellular events in fracture repair.

Bone repair consists of inflammation, intramembranous ossification, chondrogenesis, endochondral ossification, and remodeling. To better understand the translational regulation of these distinct but interrelated cellular events, we used the second generation of BD Clontechtrade mark Antibody Microarray to dissect and functionally characterize proteins differentially expressed between intact and fractured rat femur at each of these cellular events. Genetic network analysis showed that proteins differentially expressed within a given cellular event tend to be physically or functionally correlated. Seventeen such interacting networks were established over five cellular events that were most frequently associated with cell cycle, cell death, cell-to-cell signaling and interaction, and cell growth and proliferation. Eighteen molecular pathways were significantly enriched during the bone repair process, of which ERK/MAPK, NF-kB, PDGF, and T-cell receptor signaling pathways were significant during three or more cellular events. The analyses revealed dynamic temporal expression patterns and cellular-event-specific functions. The inflammation event on Day 1 was characteristic of the cell cycle-related molecular changes. The relative quiet stage of intramembranous ossification on Day 4 and the molecularly most active stage of chondrogenesis on Day 7 were featured by coordinated cell death and cell-proliferation signals. Endochondral ossification on Day 14 experienced a clear transition from the molecular/cellular function to the physiological system development/function. The osteoclast-mediated remodeling on Day 28 was highlighted by the integrin signaling pathway. The distinct changes in protein expression during these cellular events provide a molecular basis for developing cellular event-targeted therapeutic strategy to accelerate bone healing.

Animals↗

PubMed2006

The non-proteolytically active thrombin peptide TP508 stimulates angiogenic sprouting.

Thrombin is a serine protease that promotes platelet aggregation, blood coagulation, and tissue repair. A peptide derived from a non-proteolytically active region of thrombin, TP508, also promotes tissue repair and increased vascularity, yet does not activate platelet and inflammatory cascades. TP508 binds to cells with high affinity and stimulates cells independent of the proteolytically active thrombin receptors (PARs) and thus is considered to activate a non-proteolytically active receptor (non-PAR) pathway. Using a model of angiogenic sprouting, we further defined the angiogenic potential of TP508 and investigated the role of non-proteolytic, thrombin-mediated pathways in angiogenesis. The assay involves measuring angiogenic sprouting from cultured, intact microvessel fragments. In this assay, TP508 stimulated angiogenic sprouting to an extent similar to or greater than the potent angiogenic factor, VEGF. However, TP508 had no significant effect on the number of sprouts that formed per vessel. In contrast to TP508, proteolytically active receptor agonists had no effect or inhibited angiogenic sprouting. The increased sprouting activity stimulated by TP508 was VEGF dependent but did not involve an increase in VEGF mRNA expression above baseline levels. These results suggest that TP508 acts early in angiogenesis and directly on microvascular cells to accelerate sprouting, but not to induce more sprouting, in a manner different than the intact thrombin molecule.

Animals↗

PubMed2006

Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair.

Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.

Animals↗

PubMed2006

Thrombin peptide TP508 stimulates cellular events leading to angiogenesis, revascularization, and repair of dermal and musculoskeletal tissues.

The thrombin peptide, TP508, also known as Chrysalin (OrthoLogic, Tempe, Arizona), is a twenty-three-amino-acid peptide that represents a portion of the receptor-binding domain of the native human thrombin molecule that has been identified as the binding site for a specific class of receptors on fibroblasts and other cells. Preclinical studies with this peptide have shown that it can accelerate tissue repair in both soft and hard tissues by mechanisms that appear to involve up-regulation of genes that initiate a cascade of healing events. These events include recruitment and activation of inflammatory cells, directed migration of cells (chemotaxis), cell proliferation, elaboration of extra-cellular matrix, and accelerated revascularization of the healing tissues. Early preclinical dermal wound-healing studies showed that TP508 accelerated healing of both incisional wounds and full-thickness excisional wounds in normal and ischemic skin. In all of these studies, the accelerated healing was associated with increased neovascularization across the incision or in the granulating wound bed. Studies in a rat fracture model have also shown that TP508 accelerates the rate of fracture repair. Gene array analysis of fracture callus from control and TP508-treated fractures indicated that TP508 treatment was associated with an up-regulation of early response elements, inflammatory mediators, and genes related to angiogenesis. Similar to what had been seen in dermal wounds, histology from rat fracture callus twenty-one days after treatment indicated that fractures treated with TP508 had significantly more large functional blood vessels than did fractures in the control animals. In vitro studies support these in vivo data and indicate that TP508 may have a direct angiogenic effect by promoting the rate of new vessel growth. The results from phase-1 and phase-2 human clinical studies have shown a positive stimulatory effect of TP508 in the healing of diabetic ulcers and in the repair of fractures to the distal aspect of the radius. Collectively, these studies suggest that TP508 accelerates tissue repair by initiating a cascade of events that lead to an increased rate of tissue revascularization and regeneration.

Animals↗

PubMed2005

Early signals for fracture healing.

Fracture healing requires the cooperation of multiple molecular signaling pathways. To better understand this cascade of transcriptional events, we compared the gene expression profiles between intact bone and fractured bone at days 1, 2, and 4 using a rat femur model of bone healing. Cluster analysis identified several groups of genes with dynamic temporal expression patterns and stage-specific functions. The immediate-response genes are highlighted by binding activity, transporter activity, and energy derivation. We consider these activities as critical signals for initiation of fracture healing. The continuously increased genes are characterized by those directly involved in bone repair, thus, representing bone specific forefront workers. The constantly upregulated genes tend to regulate general cell growth and are enriched with genes that are involved in tumorigenesis, suggesting common pathways between two processes. The constantly downregulated genes predominantly involve immune response, the significance of which remains for further investigation. Knowledge acquired through this analysis of transcriptional activities at the early stage of bone healing will contribute to our understanding of fracture repair and bone-related pathological conditions.

Animals↗

PubMed2005

An orientation distribution function for trabecular bone.

We describe a new method for quantifying the orientation of trabecular bone from three-dimensional images. Trabecular lattices from five human vertebrae were decomposed into individual trabecular elements, and the orientation, mass, and thickness of each element were recorded. Continuous functions that described the total mass (M(phi,theta)) and mean thickness (tau(phi,theta)) of all trabeculae as a function of orientation were derived. The results were compared with experimental measurements of the elastic modulus in three principal anatomic directions. A power law scaling relationship between the anisotropies in mass and elastic modulus was observed; the scaling exponent was 1.41 (R2=0.88). As expected, the preponderance of trabecular mass was oriented along the cranial-caudal direction; on average, there was 3.4 times more mass oriented vertically than horizontally. Moreover, the vertical trabeculae were 30% thicker, on average, than the horizontal trabeculae. The vertical trabecular thickness was inversely related to connectivity (R2=0.70; P=0.07), suggesting a possible organization into either few, thick trabeculae or many thin trabeculae. The method, which accounts for the mechanical connectedness of the lattice, provides a rapid way to both visualize and quantify the three-dimensional organization of trabecular bone.

Aged↗

PubMed2005

Bone formation is enhanced by thrombin-related peptide TP508 during distraction osteogenesis.

The thrombin-related peptide, TP508, has been shown to promote soft tissue healing and fracture repair. One possible clinical application of TP508 is to accelerate bone regeneration during distraction osteogenesis, which is a lengthy procedure involving significant complications. In this study, we tested the ability of TP508 to accelerate the consolidation phase of distraction osteogenesis in a rabbit model of leg lengthening. Twenty-three rabbits had left tibiae lengthened for 1 cm over a period of 6 days. TP 508 (0, 30 and 300 microg in 300 microl saline) was injected into the distraction gaps at the beginning and the end of the lengthening phase, and all the animals were killed 2 weeks after lengthening. By the end of experiment, more animals in the TP508 treated groups had complete bony union of the distraction gaps when compared to the saline treated group. pQCT examination of the regenerates demonstrated a significantly greater bone mineral density (BMD) in the TP508 treated groups relative to the saline control group, but no statistical difference in the BMD was found between the two dosages of TP508. Bone consolidation and bone remodeling was far advanced in the TP508 300 microg treated group, and the regenerates mainly consisted of well-vascularized woven bone. In contrast, in the group that received the 30 microg TP508 treatment, focal bone defects and discontinuities of the new cortices were evident in some but not all animals. In the saline control group a majority of the animals showed large amounts of fibrous and cartilaginous tissues in the regenerates, and none of the regenerates had completed consolidation. This study has demonstrated that local application of TP508 enhanced bone formation and consolidation during distraction osteogenesis in the rabbit. The findings indicate that TP508 may be useful in promoting osteogenesis in situations when augmentative treatment for bone formation and consolidation are needed.

Animals↗

PubMed2004

Thrombin peptide (TP508) promotes fracture repair by up-regulating inflammatory mediators, early growth factors, and increasing angiogenesis.

Previous studies have shown that a single injection of thrombin peptide (TP508) accelerates fracture repair in a closed rat femoral fracture model. The present study was conducted to elucidate the molecular mechanisms of TP508 action using Affymetrix genome-scale profiling and to link early gene expression changes to fracture histology and bone strength changes. Treatment of femoral fractures with TP508 accelerated fracture repair as determined by destructive torsion testing. Blinded histological analysis demonstrated that TP508-treated fracture callus had a significant increase in blood vessels relative to the controls. Gene array analysis showed that TP508 significantly induced expression of early growth factors, inflammatory response modifiers, and angiogenesis-related genes. This study therefore suggests that TP508 promotes fracture repair through a mechanism that involves an increased induction of a number of growth factors, enhanced expression of inflammatory mediators, and angiogenesis-related genes.

Animals↗

PubMed2004

Repair of rabbit segmental defects with the thrombin peptide, TP508.

The synthetic peptide, TP508 (Chrysalin), was delivered to rabbit segmental bone defects in biodegradable controlled-release PLGA microspheres to determine its potential efficacy for enhancing healing of non-critically and critically sized segmental defects. Non-critically sized radial defects were created in the forelimbs of New Zealand White rabbits, which were randomized into three treatment groups receiving 10, 50 and 100 microg doses of TP508 in the right radius and control microspheres (without TP508) in the left radius. Torsional testing of the radii at six weeks showed a significant increase in ultimate torque, failure torque, ultimate energy, failure energy, and stiffness when treated with TP508 compared to controls (p<0.01 for all measures). Thus, TP508 appeared to enhance or accelerate bone growth in these defects. In a second set of experiments, critically sized ulnar defects were created in the forelimbs of New Zealand White rabbits, which were randomized into two groups with each rabbit receiving microspheres with 100 or 200 microg of TP508 into the right ulnar defect and control microspheres (without TP508) alone into the left ulnar defect. Bone healing was evaluated with plain radiographs, synchrotron-based microtomography, and mechanical testing. Radiographs of the rabbit limbs scored by three blinded, independent reviewers demonstrated a significantly higher degree of healing when treated with TP508 than their untreated control limbs (p<0.05). Three-dimensional synchrotron tomography of a limited number of samples showed that the new bone in TP508-treated samples had a less porous surface appearance and open marrow spaces, suggesting progression of bone remodeling. Torsional testing of the ulnae at nine weeks showed a significant increase in maximum torque and failure energy when treated with TP508 compared to controls (p<0.01 for both measures). These results suggest that TP508 in a controlled release delivery vehicle has the potential to enhance healing of segmental defects in both critically and non-critically sized defects.

Animals↗

PubMed2002

Combined magnetic fields accelerate and increase spine fusion: a double-blind, randomized, placebo controlled study.

STUDY DESIGN: The clinical study conducted was a prospective, randomized, double-blind, placebo-controlled trial. OBJECTIVES: The purpose of this study was to evaluate the effect of combined magnetic fields on the healing of primary noninstrumented posterolateral lumbar spine fusion. SUMMARY OF BACKGROUND DATA: Combined magnetic fields, a new type of biophysical stimulus, have been shown to act by stimulating endogenous production of growth factors that regulate the healing process. This is the first placebo-controlled study to assess the effect of an electromagnetic stimulus on primary noninstrumented posterolateral lumbar spine fusion surgery as well as the first evaluation of combined magnetic fields as an adjunctive stimulus to lumbar spine fusion. METHODS: This multicenter investigational study was conducted at 10 clinical sites under an Investigational Device Exemption from the United States Food and Drug Administration. Eligible patients had one-level or two-level fusions (between L3 and S1) without instrumentation, either with autograft alone or in combination with allograft. The combined magnetic field device used a single posterior coil, centered over the fusion site, with one 30-minute treatment per day for 9 months. Randomization was stratified by site and number of levels fused. Evaluation was performed 3, 6, and 9 months after surgery and 3 months after the end of treatment. The primary endpoint was assessment of fusion at 9 months, based on radiographic evaluation by a blinded panel consisting of the treating physician, a musculoskeletal radiologist, and a spine surgeon. RESULTS: Of 243 enrolled patients, 201 were available for evaluation. Among all patients with active devices, 64% healed at 9 months compared with 43% of patients with placebo devices: a significant difference (P = 0.003 by Fisher's exact test). Stratification by gender showed fusion in 67% of women with active devices, compared with 35% of those with placebo devices (P = 0.001 by Fisher's exact test). By contrast, there was not a statistically significant effect of the active device in this male study population. In the overall population of 201 patients, repeated measures analyses of fusion outcomes (by generalized estimating equations) showed a main effect of treatment, favoring the active treatment (P = 0.030). In a model with main effect and a time by treatment interaction, the latter was significant (P = 0.024), indicating acceleration of healing. Performed in the full sample of 243 patients, results of the intent-to-treat analysis were qualitatively the same as in the evaluable sample of 201 patients. DISCUSSION: This investigational study demonstrates that combined magnetic field treatment of 30 min/d increases the probability of successful spine fusion, and statistical analysis using the generalized estimating equations model suggests an acceleration of the healing process. This is the first randomized clinical trial of noninstrumented primary posterolateral lumbar spine fusion, with evaluation by a blinded, unbiased panel. This is the first double-blind study performed to date assessing noninstrumented fusion outcome with extremely critical radiographic criteria. The lower overall fusion rates in this study are attributed to the high-risk patient group with an average age of 57 years, the use of noninstrumented technique with posterolateral fusion only, and the reliance on extremely critical radiographic and clinical criteria and blinded panel for fusion assessment without surgical confirmation. CONCLUSIONS: In conclusion, the adjunctive use of the combined magnetic field device was statistically beneficial in the overall patient population, as has been shown in previous studies of adjunctive bone growth stimulation for spine fusion. For the first time, stratification of fusion success data by gender demonstrated that the female study population responded positively to the adjunctive combined magnetic field treatment, with no statistically significant effect observed in the male study population. Adjunctive use of the combined magnetic field device significantly increased the 9-month success of radiographic spinal fusion and showed an acceleration of the healing process.

Adult↗