James T. Ryaby

Thrombin related peptide TP508 promoted fracture repair in a mouse high energy fracture model

BackgroundThrombin related peptide (TP508) is a 23 amino-acid synthetic peptide that represents a portion of the receptor-binding domain of thrombin molecule. Previous studies have shown that TP508 can accelerate musculoskeletal tissue repair including fracture healing.ObjectivesThe aim of this study was to investigate the effect of TP508 on fracture healing in a murine fracture model representing high energy fracture situation.MethodsEighty CD 1 mice underwent controlled quadriceps muscle crush and open transverse mid diaphyseal femoral fracture that was then fixed with an external fixator. Animals were randomised into four groups to receive an intra-operative dose of either 100 μg TP508 into the fracture gap; 100 μg TP508 into the surrounding damaged muscle tissues; 10 μg TP508 into the fracture gap, or control equal amount of saline into the fracture gap. Radiographic assessment was performed weekly for 5 weeks; histological analysis was at 3 and 5 weeks post fracture and biomechanical testing of the fractured bone was performed at 5 weeks post fracture.ResultsMechanical testing data showed that the fracture stiffness was significantly higher in the group receiving 100 μg TP508 into the fracture gap than other groups. Histological and radiographic analysis revealed a trend of increase in bone formation in the 100 μg TP508 injected into the fracture gap group compared to the saline control group. It was noted that the scar tissues was significantly less in Group II comparing with the saline control group and there was increased blood vessel formation in the crushed muscles and fracture gap areas in the groups receiving TP508 comparing to the saline control group.ConclusionThe results from this study demonstrated the use of thrombin related peptide TP508 in the situation of a high energy fracture can promote fracture healing and reduce the potential complications such as muscle fibrosis and fracture delayed or non-union.

Thrombin peptide Chrysalin® stimulates healing of diabetic foot ulcers in a placebo‐controlled phase I/II study

Thrombin and thrombin peptides play a role in initiating tissue repair. The potential safety and efficacy of TP508 (Chrysalin®) treatment of diabetic foot ulcers was evaluated in a 60‐subject, prospective, randomized, double‐blind, placebo‐controlled phase I/II clinical trial. Chrysalin® in saline or saline alone was applied topically, twice weekly, to diabetic ulcers with standardized care and offloading. A dose‐dependent effect was seen in the per‐protocol population where 1 and 10 μg Chrysalin® treatment resulted in 45 and 72% more subjects with complete healing than placebo treatment. Chrysalin® treatment of foot ulcers more than doubled the incidence of complete healing (p<0.05), increased mean closure rate ∼80% (p<0.05), and decreased the median time to 100% closure by ∼40% (p<0.05). Chrysalin® treatment of heel ulcers within this population resulted in mean closure rates 165% higher than placebos (p<0.02) and complete healing in 86% (6/7) of ulcers compared with 0% (0/5) of placebo ulcers (p<0.03). Local wound reactions and adverse events (AEs) were equal between groups with no reported drug‐related changes in laboratory tests or serious AEs. These results indicate the potential safety and efficacy of Chrysalin® for treatment of diabetic foot ulcers.

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.

Inhibition of Phosphate-Induced Apoptosis in Resting Zone Chondrocytes by Thrombin Peptide 508

Growth plate chondrocytes are susceptible to apoptosis. Terminally differentiated chondrocytes are deleted via apoptosis, which primes the growth plate to vascular invasion and subsequent bone formation. Whether less differentiated resting zone chondrocytes are subject to the same mechanism that governs the apoptotic pathway of more differentiated growth zone chondrocytes is not known. In our current study, we demonstrated that inorganic phosphate, a key inducer of growth plate chondrocyte apoptosis, also causes apoptosis in resting zone chondrocytes, via a pathway similar to the one in growth zone chondrocytes. Our results demonstrated that the conditions that cause growth plate chondrocyte apoptosis lie in the external environment, instead of the differences in differentiation state.

Effects of pulsed electromagnetic field therapy at different frequencies and durations on rotator cuff tendon-to-bone healing in a rat model

BACKGROUND Rotator cuff tears affect millions of individuals each year, often requiring surgical intervention. However, repair failure remains common. We have previously shown that pulsed electromagnetic field (PEMF) therapy improved tendon-to-bone healing in a rat rotator cuff model. The purpose of this study was to determine the influence of both PEMF frequency and exposure time on rotator cuff healing. METHODS Two hundred ten Sprague-Dawley rats underwent acute bilateral supraspinatus injury and repair followed by either Physio-Stim PEMF or high-frequency PEMF therapy for 1, 3, or 6 hours daily. Control animals did not receive PEMF therapy. Mechanical and histologic properties were assessed at 4, 8, and 16 weeks. RESULTS Improvements in different mechanical properties at various endpoints were identified for all treatment modalities when compared with untreated animals, regardless of PEMF frequency or duration. Of note, 1 hour of Physio-Stim treatment showed significant improvements in tendon mechanical properties across all time points, including increases in both modulus and stiffness as early as 4 weeks. Collagen organization improved for several of the treatment groups compared with controls. In addition, improvements in type I collagen and fibronectin expression were identified with PEMF treatment. An important finding was that no adverse effects were identified in any mechanical or histologic property. CONCLUSIONS Overall, our results suggest that PEMF therapy has a positive effect on rat rotator cuff healing for each electromagnetic fundamental pulse frequency and treatment duration tested in this study.

Role of pulsed electromagnetic fields (PEMF) on tenocytes and myoblasts—potential application for treating rotator cuff tears

The post‐surgery integrity of the tendons and muscle quality are the two major factors in success of rotator cuff (RC) repair. Though surgical techniques for rotator cuff repair have significantly improved in the past two decades, there are no effective treatments to improve tendon‐to‐bone healing and muscle quality after repair at this point in time. Pulsed electromagnetic fields (PEMF) have previously been used for promoting fracture healing. Previous studies have shown that PEMF has a positive role in promoting osteoblast precursors proliferation and differentiation. However, PEMFs effect on tenocytes and muscle cells has not been determined fully yet. The purpose of this study is to define the role of a commercially available PEMF on tenocytes and myoblasts growth and differentiation in vitro. Human rotator cuff tenocytes and C2C12 murine myoblasts were cultured and treated with PEMF for 2 weeks under regular and inflammatory conditions. Our results showed that 2 weeks treatment of PEMF enhanced gene expressions of growth factors in human rotator cuff tenocytes under inflammatory conditions. PEMF significantly enhanced C2C12 myotube formation under normal and inflammatory conditions. Results from this study suggest that PEMF has a positive role in promoting tenocyte gene expression and myoblast differentiation. Therefore, PEMF may potentially serve as a non‐operative treatment to improve clinical incomes rotator cuff tendon repairs. Results

Pulsed electromagnetic field therapy improves tendon‐to‐bone healing in a rat rotator cuff repair model

Rotator cuff tears are common musculoskeletal injuries often requiring surgical intervention with high failure rates. Currently, pulsed electromagnetic fields (PEMFs) are used for treatment of long‐bone fracture and lumbar and cervical spine fusion surgery. Clinical studies examining the effects of PEMF on soft tissue healing show promising results. Therefore, we investigated the role of PEMF on rotator cuff healing using a rat rotator cuff repair model. We hypothesized that PEMF exposure following rotator cuff repair would improve tendon mechanical properties, tissue morphology, and alter in vivo joint function. Seventy adult male Sprague–Dawley rats were assigned to three groups: bilateral repair with PEMF (n = 30), bilateral repair followed by cage activity (n = 30), and uninjured control with cage activity (n = 10). Rats in the surgical groups were sacrificed at 4, 8, and 16 weeks. Control group was sacrificed at 8 weeks. Passive joint mechanics and gait analysis were assessed over time. Biomechanical analysis and μCT was performed on left shoulders; histological analysis on right shoulders. Results indicate no differences in passive joint mechanics and ambulation. At 4 weeks the PEMF group had decreased cross‐sectional area and increased modulus and maximum stress. At 8 weeks the PEMF group had increased modulus and more rounded cells in the midsubstance. At 16 weeks the PEMF group had improved bone quality. Therefore, results indicate that PEMF improves early tendon healing and does not alter joint function in a rat rotator cuff repair model.

Pulsed electromagnetic field (PEMF) treatment reduces expression of genes associated with disc degeneration in human intervertebral disc cells

BACKGROUND CONTEXT Pulsed electromagnetic field (PEMF) therapies have been applied to stimulate bone healing and to reduce the symptoms of arthritis, but the effects of PEMF on intervertebral disc (IVD) biology is unknown. PURPOSE The purpose of this study was to determine how PEMF affects gene expression of IVD cells in normal and inflammatory environments. STUDY DESIGN/SETTING This was an in vitro human cell culture and microarray gene expression study. METHODS Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were separately encapsulated in alginate beads and exposed to interleukin 1α (IL-1α) (10 ng/mL) to stimulate the inflammatory environment associated with IVD degeneration and/or stimulated by PEMF for 4 hours daily for up to 7 days. RNA was isolated from each treatment group and analyzed via microarray to assess IL-1α- and PEMF-induced changes in gene expression. RESULTS Although PEMF treatment did not completely inhibit the effects of IL-1α, PEMF treatment lessened the IL-1α-induced upregulation of genes expressed in degenerated IVDs. Consistent with our previous results, after 4 days, PEMF tended to reduce IL-1α-associated gene expression of IL-6 (25%, p=.07) in NP cells and MMP13 (26%, p=.10) in AF cells. Additionally, PEMF treatment significantly diminished IL-1α-induced gene expression of IL-17A (33%, p=.01) and MMP2 (24%, p=.006) in NP cells and NFκB (11%, p=.04) in AF cells. CONCLUSIONS These results demonstrate that IVD cells are responsive to PEMF and motivate future studies to determine whether PEMF may be helpful for patients with IVD degeneration.

Dynamic imaging demonstrates that pulsed electromagnetic fields (PEMF) suppress IL-6 transcription in bovine nucleus pulposus cells†

Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Pulsed electromagnetic fields (PEMF) are noninvasive biophysical stimulus that has been used extensively in the orthopaedic field for many years. However, the specific cellular responses and mechanisms involved are still unclear. The objective of this study was to assess the time‐dependent PEMF effects on pro‐inflammatory factor IL‐6 expression in disc nucleus pulposus cells using a novel green fluorescence protein (GFP) reporter system. An MS2‐tagged GFP reporter system driven by IL‐6 promoter was constructed to visualize PEMF treatment effect on IL‐6 transcription in single living cells. IL‐6‐MS2 reporter‐labeled cells were treated with IL‐1α to mimic the in situ inflammatory environment of degenerative disc while simultaneously exposed to PEMF continuously for 4 h. Time‐lapse imaging was recorded using a confocal microscope to track dynamic IL‐6 transcription activity that was demonstrated by GFP. Finally, real‐time RT‐PCR was performed to confirm the imaging data. Live cell imaging demonstrated that pro‐inflammatory factor IL‐1α significantly promoted IL‐6 transcription over time as compared with DMEM basal medium condition. Imaging and PCR data demonstrated that the inductive effect of IL‐1α on IL‐6 expression could be significantly inhibited by PEMF treatment in a time‐dependent manner (early as 2 h of stimulus initiation). Our data suggest that PEMF may have a role in the clinical management of patients with chronic low back pain. Furthermore, this study shows that the MS2‐tagged GFP reporter system is a useful tool for visualizing the dynamic events of mechanobiology in musculoskeletal research.

Mechanical Stress on Suspended Cortical Bone Sample by Low Frequency Magnetic Field

Low-frequency (LF), low-amplitude alternating current (ac) magnetic fields are approved by the U.S. Food and Drug Administration as an adjunct to the orthopedic surgery procedure (spinal fusion surgery) and for the treatment of selected bone healing complications (fracture non-union). The biological effects are understood to rely on a contact-less induction of electric currents in the electrically conducting compartments of the bone. Here, we investigate another potential mechanism of the LF magnetic field effect on bone by measuring mechanical force on bone due to its diamagnetic susceptibility. The biological rationale is the bones exquisite sensitivity to local mechanical stresses that determine its structure and physiology. Thus, the periodic magnetic microstress effects on bone over an extended period of the typical clinical LF magnetic field application (hours daily over a period of a few months) could contribute to bone regeneration. The aim was to demonstrate the diamagnetic field effect on thin, human cortical bone slice preparations (from commercial tissue repository) suspended in the vicinity of a rotating cylindrical, diametrically magnetized permanent magnet. The data provide a basis for the calculation of diamagnetic microstresses generated by the clinical LF magnetic field stimulation devices and their significance as potential contributing factors to the biological mechanism of action of LF ac magnetic fields.