Lorne Beckman

Effects of osteogenic protein-1 on distraction osteogenesis in rabbits

In this study we tested the effect of locally applied osteogenic protein 1 (OP-1) on distraction osteogenesis in rabbits. Seven days after tibial osteotomy, distraction was started at a rate of 0.25 mm per 12 h for 3 weeks. At the end of the distraction period, OP-1 was injected at the site of osteotomy. Four different dosages were tested (0, 80, 800, or 2000 microg; eight rabbits per dose group). Rabbits were sacrificed 3 weeks later, and histologic, densitometric, and biomechanical parameters were assessed. No significant differences were found between groups for any parameter. To explain why this approach was only modestly successful, the expression of BMP receptor protein in the newly formed tissue was analyzed by immunohistochemistry. Strong expression of BMP receptor IA, IB, and II was found during the early distraction phase, but not during later stages of the process. Thus, it appears that the lack of receptor protein in the target tissue impairs the effect of OP-1 given at the end of the distraction period. Possibly, OP-1 could be more useful when applied early in the distraction phase.

Evaluation of quantitative magnetic resonance imaging, biochemical and mechanical properties of trypsin‐treated intervertebral discs under physiological compression loading

To investigate the influence of targeted trypsin digestion and 16 hours compression loading on MR parameters and the mechanical and biochemical properties of bovine disc segments.

Physiological loading can restore the proteoglycan content in a model of early IVD degeneration.

A hallmark of early IVD degeneration is a decrease in proteoglycan content. Progression will eventually lead to matrix degradation, a decrease in weight bearing capacity and loss of disc height. In the final stages of IVD degradation, fissures appear in the annular ring allowing extrusion of the NP. It is crucial to understand the interplay between mechanobiology, disc composition and metabolism to be able to provide exercise recommendations to patients with early signs of disc degeneration. This study evaluates the effect of physiological loading compared to no loading on matrix homeostasis in bovine discs with induced degeneration. Bovine discs with trypsin-induced degeneration were cultured for 14 days in a bioreactor under dynamic loading with maintained metabolic activity. Chondroadherin abundance and structure was used to confirm that a functional matrix was preserved in the chosen loading environment. No change was observed in chondroadherin integrity and a non-significant increase in abundance was detected in trypsin-treated loaded discs compared to unloaded discs. The proteoglycan concentration in loaded trypsin-treated discs was significantly higher than in unloaded disc and the newly synthesised proteoglycans were of the same size range as those found in control samples. The proteoglycan showed an even distribution throughout the NP region, similar to that of control discs. Significantly more newly synthesised type II collagen was detected in trypsin-treated loaded discs compared to unloaded discs, demonstrating that physiological load not only stimulates aggrecan production, but also that of type II collagen. Taken together, this study shows that dynamic physiological load has the ability to repair the extracellular matrix depletion typical of early disc degeneration.

Topical application of complement C3 in collagen formulation increases early wound healing.

Abstract Background: The complement system is composed of bactericidal and hemolytic proteins that increase capillary leakage and inflammatory cell migration. The role of complement C3 to augment wound healing has not yet been studied. Methods: We examined the effects of topical complement C3 formulation at two concentrations (10 and 100 nM) on the rat surgical skin incision model. Skin was examined for maximal breaking strength and sectioned for histological examination. Fibronectin and collagen I content were measured using western blot analysis. Results: There was a statistically significant 74% increase in maximum wound strength with the topical application of 100 nM of C3 at day 3 (850 ± 138 g) when compared to the control rats (490 ± 57 g). Histological correlation was seen with an increased inflammatory cell and fibroblast infiltration in treated wounds as compared to control rats as early as 3 days post-wounding. Western blots revealed increased fibronectin and collagen I levels in C3 treated wounds. Conclusions: Topical application of complement C3 in collagen formulation to skin wounds significantly increases wound healing as early as 3 days after wounding. This is correlated with increased inflammatory cell recruitment and the subsequent early fibroblast migration and increased collagen deposition and organization in wounds.

Accelerated wound healing with topical application of complement C5.

Background: Delayed-healing traumatic, surgical, and chronic wounds can be detrimental to patients and the health care system. The authors set out to investigate the effects of complement C5, a naturally occurring chemotactic cytokine, on wounds. Methods: The authors examined the effects of complement C5 on the rat paired skin incision model. Each rat served as its own control where topical collagen was applied to one incision and 100 nM of C5 in collagen vehicle was applied to the other incision. Rats were killed on days 3 (n = 6), 7 (n = 6), and 28 (n = 5) after wounding. Results: There was a statistically significant, 65 percent increase in maximum wound breaking strength with the topical application of C5 at day 3 (p < 0.01). The increase persisted to 14 percent at 7 days after wounding (p < 0.05). When compared with the sham group, the C5-treated wound strength increased by 83 percent at day 3 and 64 percent at day 7. There was no change in breaking strength at 28 days. Western blot analysis demonstrated a significant increase in collagen and fibronectin content in the C5-treated wounds. Conclusions: Topical application of C5 to skin wounds significantly increases wound healing maximum breaking strength as early as 3 days and up to 7 days after wounding. C5 accelerated wound healing by at least 4 days in the first week of wounding. This was correlated with an increase in vascular permeability, increased inflammatory cell recruitment, subsequent fibroblast migration, and increased collagen deposition.

Complements c3 and c5 individually and in combination increase early wound strength in a rat model of experimental wound healing.

Background. Complements C3 and C5 have independently been shown to augment and increase wound healing and strength. Our goal was to investigate the combinatorial effect of complements C3 and C5 on wound healing. Methods. Each rat served as its own control where topical collagen was applied to one incision and 100 nM of C3 and C5 in collagen vehicle was applied to the other incision (n = 6). To compare between systemic effects, a sham group of rats (n = 6) was treated with collagen alone on one wound and saline on the other. At day 3, the tissue was examined for maximal breaking strength (MBS) and sectioned for histological examination. Results. There was a statistically significant 88% increase in MBS with the topical application of C3C5 when compared to sham wounds (n < 0.05). This was correlated with increased fibroblast and collagen deposition in the treated wounds. Furthermore, there appeared to be an additive hemostatic effect with the C3C5 combination. Conclusions. The combination of complements C3 and C5 as a topical application drug to skin wounds significantly increased wound healing maximum breaking strength as early as 3 days.

The effects of topical collagen treatment on wound breaking strength and scar cosmesis in rats.

BACKGROUND Topical application of collagen has been suggested to enhance wound healing; however, its long-term effect on wounds has not been studied in a rat model. HYPOTHESIS Topical application of collagen type I will not facilitate incision healing or cosmesis in rats up to 28 days postwounding. METHODS The effects of bovine collagen type I (6 mg/mL) on the rat surgical paired skin incision model were examined. Each rat served as its own control in which topical collagen was applied to one incision while normal saline (0.9%) was applied to the other incision. Rats were euthanized three (n=6), seven (n=6) and 28 (n=5) days after wounding. Tissue harvested from each time point was examined for maximal breaking strength, and for biochemical and histological analysis. RESULTS There were no statistically significant differences (ie, P<0.05) in maximum wound breaking strength between the collagen- and saline-treated wounds at all time points. Histological analysis revealed a similar infiltration of inflammatory cells and fibroblasts in the wound edges of all incisions when matched with time of wounding. Western blot analysis revealed no differences in fibronectin or collagen I content in all wounds in each rat. CONCLUSIONS The topical application of collagen did not facilitate wound healing from three to 28 days in the rat wound model.

Cadaveric device-injected very high-viscosity cement during vertebroplasty

Introduction: Cement extravasation during vertebroplasty (VP) is the most commonly reported complication. Cement viscosity is considered the single most important predictor of the risk of extravasation. Certainly, injecting high-viscosity cement (HVC) is difficult to utilize in real practice. We invented a new device capable of injecting high-viscosity with ease and at a distance to avoid radiation. The aim of this study is to confirm the efficacy and safety of the new device on cadaveric vertebrae. Methodology: A 126 osteoporotic vertebral bodies were harvested from cadavers. Eighty vertebrae were included in the study. Computer-randomization software was used to allocate specimens over two main groups, Conventional VP and New Device. Both groups were further subdivided into two subgroups; high-viscosity and low-viscosity. A custom device was used on each vertebra to induce a compression fracture. Results: Injecting HVC was associated with a lower leakage volume compared with low-viscosity cement. HVC was associated with no leakage into the spinal canal. It was also associated with a low incidence of vascular extravasation (P < 0.001). The mean volume of cement leakage in the low-viscosity group was 0.23 and 0.15 cc, for the Conventional VP and New Device, respectively. In both groups, the most common site for leakage was the vertebral end plate, which was exhibited more in the low-viscosity group (71.5%) compared with the high-viscosity group (42.5%). The preset target amount of cement to be injected was reached in 99% of the time when injecting HVC with the New Device, compared with 62% using the Conventional VP. In both groups, there was no correlation between the amount of cement injected and the amount of leakage. Conclusion: The new device is capable of injecting HVC easily, with a lower incidence of cement leakage. It also minimized the risk of radiation exposure to the surgeon.

Vertebroplasty Injections Using High Viscosity Cement: a Cadaveric Study

Introduction Vertebral compression fracture (VCF) is a unique type of fractures involving the body of vertebrae. It is associated with a significant decline in functionality and poor quality of life. Osteoporosis, or decreased bone density, is one of the leading causes of VCF. It is commonly seen amongst elderly, especially in post-menopausal women. Vertebroplasty (VP) is a minimally invasive percutaneous technique that involves injecting cement into a pathologic vertebral body, with the aim of pain relief and deformity correction. It is considered the best next line of management when conservative management fails to control symptoms. Although considered minimally invasive, VP has ~1 to 5% risk of clinically evident complications. Where, cement extravasation is the most commonly reported in the literature. Cement viscosity is considered the single most important predictor of the risk of extravasation. Unfortunately, injecting high viscosity cement is difficult to utilize in real practice. We invented a new device capable of injecting high viscosity with ease and at a distance to avoid radiation. The aim of this study is to confirm the efficacy and safety of the new device on cadaveric vertebrae. Materials and Methods For this prospective case-control cohort study, 126 osteoporotic vertebral bodies were harvested form 14 fresh whole human cadavers. DEXA scan and simple X-rays were performed on all specimens to confirm the diagnosis of osteoporosis, and to rule out any pre-existing fractures. A total of 80 vertebral were included in the study. Computer-randomization software was used to allocate specimens over two main groups, Conventional VP and New Device. Both groups were further subdivided into two subgroups, High-Viscosity and Low-Viscosity. A custom device was used on each vertebra to induce a compression fracture. All augmentations were done following the same method commonly practiced in clinical treatment. X-ray radiographs were used to measure and compare cement distribution within the body between groups. Results Injecting high viscosity cement was associated with a lower leakage volume compared with low viscosity cement, with a mean of 0.06cc for the Conventional VP and 0.08cc with the New Device. High viscosity cement was associated with no leakage into the spinal canal. It was also associated with a low incidence of vascular extravasation (p < 0.001). The mean volume of cement leakage in the low viscosity group was 0.23cc and 0.15cc, for the Conventional VP and New Device respectively. In both groups, the most common site for leakage was the vertebral end plate, which was exhibited more in the low viscosity group (71.5%) compared with the high viscosity group (42.5%). The preset target amount of cement to be injected was reached in 99% of the time when injecting high viscosity cement with the New Device, compared with 62% using the Conventional VP. In both groups, there was no correlation between the amount of cement injected and the amount of leakage. Conclusion The new device is capable of injecting high viscosity cement easily, with a lower incidence of cement leakage. It also minimized the risk of radiation exposure to the surgeon.

Evaluation of Tissue Repair Using MRI Imaging of Human Intervertebral Discs Cultured in a Bioreactor

Introduction Low back pain is a major problem world-wide, affecting the quality of life for millions of people. Low back pain also has a tremendous impact on direct and indirect global healthcare costs. Intervertebral disc (IVD) degeneration has been strongly associated with low back pain. Long-term organ culture of human IVDs is essential to study IVD degeneration and repair. Using an ex vivo approach, the relationship between mechanobiology, disc matrix composition and metabolism can be better understood in the context of degenerative disease. We have developed a bioreactor where intact human discs can be cultured in a controlled dynamically loaded environment. Here, we aimed to determine the most suitable loading parameters for human discs culture by assessing IVD tissue integrity and cell viability under low, medium and high magnitude cyclic load. Furthermore, we investigated the suitability of this model toward cell supplementation strategies for tissue repair and developed a novel, single disc MRI imaging sequence aimed at direct visualization of tissue repair. Materials and Methods Human IVDs were isolated from lumbar spine segments as previously described. Spines were obtained with consent through the Transplant Quebec Organ Donation Program from individuals who had undergone sustained brain death. Discs were cultured under 3 different loading schemes to mimic a sedentary lifestyle: low 0.1–0.3, medium 0.1–0.6 and high 0.1–1.2 MPa loads. Cell viability and matrix stability was assessed following 10 days of loading. Feasibility of cell/hydrogel implantation was determined over 14 days of medium dynamic loading. To determine whether isolated discs could be imaged by MRI, extracted individual discs were visualized for T1 and T2 signals using a novel sequence using a small animal Bruker 7.5 Tesla MRI. Results Cell viability was maintained at greater than 80% throughout the discs at low and medium loads. Viability dropped to ~60–70% throughout the discs under high loads. Proteoglycan content remained stable in all loading protocols (~50 μg sGAG/mg tissue), as did CHAD and newly synthesized collagen II protein. To test for feasibility of cell therapies in the bioreactors, NP cells combined with a hydrogel were injected into discs and cultured under medium load. 14 days after dynamic culture, the injected cells were mainly localized to the NP region with greater than 90% viability. The small animal MRI was able to obtain well-defined images of isolated discs, with details of tissue integrity and proteoglycan content. Conclusion Our ex vivo model of dynamic human IVD culture can be used as a platform on which to study mechanisms of degeneration as well as for novel avenues aimed at biological repair using bioactive substances or cell based therapies. Cells and bioactive substances can be administered within hydrogels thereby enhancing the reparative properties. Furthermore, it is feasible to assess repair potential of the therapies by comparing MRI scans pre- and post-therapy.