rhBMP-6 stimulated osteoprogenitor cells enhance posterolateral spinal fusion in the New Zealand white rabbit.

BACKGROUND CONTEXT: The nonunion rate after posterolateral spinal fusion can be as high as 35%. This has stimulated interest in the development of techniques for enhancing new bone formation, including the addition of bioactive peptides or the use of cell-based therapies, including genetically modified cells. In previous studies we have demonstrated that exposing autologous, marrow-derived osteoprogenitor cells to a recombinant human bone morphogenetic protein-6 (rhBMP-6) containing extracellular matrix induces osteoblastic differentiation, and that these cells are capable of increasing new bone formation. Growth of autologous cells on a synthetic rhBMP-6 containing matrix yields a population of stimulated osteoprogenitor cells, without the expense of adding large amounts of rhBMP-6 directly, or the risks inherent in the use of genetically altered cells. PURPOSE: This study was performed to evaluate the potential of rhBMP-6 stimulated osteoprogenitor cells (stOPC) to enhance the rate and strength of posterolateral spinal fusion. STUDY DESIGN: Prospective in vivo animal study OUTCOME MEASURES: Radiographic evidence of spinal fusion, biomechanical testing of explanted spines, histological analysis of new bone formation METHODS: Single-level posterolateral spinal arthrodeses were performed in 69 New Zealand white rabbits. Autologous marrow stem cells were concentrated and then plated on an rhBMP-6-rich extracellular matrix synthesized by genetically engineered mouse C3H10T1/2 cells. Animals in Groups I (n=18) and II (n=18) received autografts of 30M and 60M rhBMP-6 stOPCs in guanidine extracted demineralized bone matrix (gDBM), respectively, whereas those in Group III (n=13) received iliac crest bone graft (ICBG). Those in Group IV (n=10) received gDBM, and those in Group V (n=10) underwent decortication only. Assessment of fusion was made with serial radiographs, manual palpation of the explanted spines, and biomechanical testing. The fusion masses from two animals each in Groups I, II, and IV were evaluated histologically. RESULTS: Fifty-three animals were available for analysis at the conclusion of the study. In these animals, the arthrodesis rate was significantly higher after treatment with rhBMP-6 stOPCs (77% for both Groups I and II by palpation) than ICBG, gDBM, or decortication alone (Group III=55%, IV=20% and V=0%, respectively). Similarly, the peak loads to failure of the fusion masses in Groups I and II (212.5+/-37.8 N and 234.6+/-45.7 N) were significantly greater than the corresponding values in the other groups (Group III=155.9+/-36.4N, Group IV=132.7+/-59.9N, and Group V=92.8+/-18.4N), though when only the fused specimens in Groups I, II, and III were compared, only Group II was significantly different than Group III (234.6+/-45.7N and 155.9+/-36.4N, respectively). The fusion masses in the rhBMP-6 stOPC-treated animals were typified by a thin, fusiform cortical shell, newly formed trabecular bone emanating from the decorticated transverse processes, and residual unremodeled gDBM carrier particles. The fusion masses in the gDBM treated bones were morphologically similar, though they contained less newly formed bone. CONCLUSIONS: The use of rhBMP-6 stOPCs in a carrier of gDBM significantly enhanced the rate and strength of single-level posterolateral spinal arthrodeses in the New Zealand white rabbit, compared with ICBG, gDBM, and decortication alone. Our results confirm that the stimulation of marrow-derived osteoprogenitor cells by growing them on a rhBMP-6 containing extracellular matrix is feasible. Further investigation is warranted to determine the relative contribution of rhBMP-6 stimulation and the number of cells implanted, as well as strategies for optimizing the technique for clinical application.     

Rectovaginal fistula model in the New Zealand white rabbit

Introduction and hypothesis  

The purpose of this study was to create an animal model to study rectovaginal fistula repair.     

Evaluation of a knitted polytetrafluoroethylene mesh placed intraperitoneally in a New Zealand white rabbit model

Background

The intraperitoneal application of surgical mesh remains a controversial issue because of possible complications, especially adhesion and fistula formation. This study aimed to assess the potential of a knitted polytetrafluoroethylene (PTFE) mesh for intraabdominal implantation.

Methods

Twenty-eight 5?×?5?cm samples of knitted macroporous PTFE mesh and light-weight polypropylene mesh (LW-PP) were implanted intraperitoneally in 14 New Zealand white rabbits in a randomized manner and fixed using eight polypropylene stitches. After 90?days, the adhesion formation, adhesion score, shrinkage, strength of fixation to the abdominal wall, and histologic biocompatibility were assessed.

Results

No intraoperative or anesthesia-related complications or mesh infection were recorded. The average area covered by adhesions was 4.7?±?7.2% for the PTFE and 36.4?±?36.1% for the LW-PP. The median adhesion score was 0 for the PTFE and 8 for the LW-PP. Shrinkage was 36.9?±?12.9% for the PTFE mesh and 12.6?±?8.72% for the LW-PP. The mesh-to-abdominal wall fixation strength was almost the same for both materials (PTFE 3.6?±?1.9 vs. LW-PP 3.6?±?2.9). The inflammatory cell count was almost the same for the two groups, with no statistically significant difference. The width of the inner granuloma was equal (PTFE 10.5?±?0.9 vs. LW-PP 11.1?±?0.9). The outer granuloma was reduced significantly in the PTFE group (PTFE 23.0?±?2.1 vs. LW-PP 33.6?±?7.9). One of the animals in the PTFE group died on postoperative day 12 because of ileus. The reason was an adhesion of the small intestine to the polypropylene fixation stitch, which caused small intestine strangulation.

Conclusions

The knitted PTFE mesh induces fewer intraperitoneal adhesions of lower density than the light-weight polypropylene mesh. The strength of the knitted PTFE mesh fixation to the abdominal wall is comparable with that of the light-weight polypropylene mesh, but the shrinkage is greater. The biocompatibility of the knitted PTFE mesh is comparable with that of the light-weight polypropylene implant.     

MAC of sevoflurane in humans and the New Zealand white rabbit

The minimum alveolar concentration of sevoflurane necessary to prevent movement in 50 per cent of patients (MAC) was determined to be 2.05 per cent in 20 adult surgical patients. Because this value was higher than the only other experimentally determined human MAC value for sevoflurane (1.71 per cent), MAC was also determined in New Zealand white rabbits. Comparisons of the MAC ratios of sevoflurane to other volatile anaesthetics in both the human and the rabbit suggest that the human MAC value we obtained for sevoflurane is consistent with experimental determinations of MAC of other volatile anaesthetics in humans.     

Biomechanical evaluation of the New Zealand white rabbit lumbar spine: a physiologic characterization

Physiologic motions of the human, sheep, and calf lumbar spines have been well characterized. The size, cost, and ease of care all make the rabbit an attractive alternative choice for an animal lumbar spine model. However, comparisons of normal biomechanical characteristics of the rabbit lumbar spine have not been made to the spines of larger species. The purpose of this study was to establish baseline physiologic kinematic data for the rabbit lumbar spine. Ten skeletally mature New Zealand white rabbit osteoligamentous spines were obtained. L4-L7 spine segments were harvested and mounted. Multi-directional flexibility testing was performed by applying pure moments up to 0.27 Nm. Resulting rotations were measured using an Optotrak system. Data were analyzed for each intervertebral level in the three planes of rotation. The three levels tested had roughly similar range of motion (ROM). The mean (SD) angular ROMs in flexion for L4-L5, L5-L6, L6-L7 were 12.10° (2.59°), 12.38° (2.70°), and 15.17° (3.22°), respectively. The ROMs in extension were 5.86° (1.21°), 5.58° (1.48°), and 6.13° (2.03°). Lateral bending and axial rotation were roughly symmetric due to the symmetric nature of the spine. For right lateral bending, the ROMs were 8.25° (2.44°), 4.96° (1.70°), and 4.25° (1.20°). For left axial rotation, the ROMs were 1.23° (1.16°), 0.35° (0.61°), 0.87° (0.64°). Neutral zone (NZ) was on average 60% (29%) of ROM for the motions studied. The physiologic ROM of the New Zealand white rabbit lumbar spine was found to be similar between the rabbit and human. This relatively conserved physiologic flexibility supports the use of the rabbit as a model of the lumbar spine for kinematic studies. However, the overall NZ was found to be a greater percentage of ROM in the rabbit than the corresponding percentage in the human (60% as compared to 25%). This suggested that the rabbit lumbar spine has a greater laxity than that of the human. Received: 23 August 1999 Revised: 15 December 1999 Accepted: 26 January 2000     

Interposition grafts for rectovaginal fistula repair in the New Zealand white rabbit

Introduction and hypothesis

The objective was to use an animal model to study different types of interposition grafts for rectovaginal fistula repair.

Methods

Twelve New Zealand white rabbits underwent surgical creation of a rectovaginal fistula, followed by repair. Four repair techniques were studied; three with interposition grafts and one control group without a graft. Animals were euthanized at 4-week intervals and underwent gross and histologic analysis.

Results

The mean rectovaginal wall thickness was greatest in the control group (5.6 mm) and thinnest in the autologous rectus fascia (4.2 mm) and porcine small intestine submucosa (5.1 mm) groups. The polypropylene graft had a mean thickness of 5.4 mm and elicited a strong, protracted inflammatory response. All fistulas were successfully closed except one porcine small intestine submucosa repair.

Conclusions

There is no benefit from interposition graft use for rectovaginal fistula repair in our New Zealand white rabbit model.     

Novel nitric oxide producing probiotic wound healing patch: preparation and in vivo analysis in a New Zealand white rabbit model of ischaemic and infected wounds

The treatment of chronic wounds poses a significant challenge for clinicians and patients alike. Here we report design and preclinical efficacy of a novel nitric oxide gas (gNO)-producing probiotic patch for wound healing. Specifically, a wound healing patch using lactic acid bacteria in an adhesive gas permeable membrane has been designed and investigated for treating ischaemic and infected full-thickness dermal wounds in a New Zealand white rabbit model for ischaemic wound healing. Kaplan-Meier survival curves showed increased wound closure with gNO-producing patch-treated wounds over 21 days of therapy (log-rank P = 0·0225 and Wilcoxon P = 0·0113). Cox proportional hazard regression showed that gNO-producing patch-treated wounds were 2·52 times more likely to close compared with control patches (hazard P = 0·0375, score P = 0·032 and likelihood ratio P = 0·0355), and histological analysis showed improved wound healing in gNO-producing patch-treated animals. This study may provide an effective, safe and less costly alternative for treating chronic wounds.     

Evaluation of intraperitoneal placement of absorbable and nonabsorbable barrier coated mesh secured with fibrin sealant in a New Zealand white rabbit model

Background  

This study aimed to evaluate the acute and chronic fixation strength of fibrin sealant (FS) as an alternative method of fixation for laparoscopic ventral hernia repair (LVHR).     

Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model

We tested the hypothesis that erythropoietin (EPO) enhances bone formation after posterolateral spinal fusion (PLF) in a rabbit model. Thirty-four adult rabbits underwent posterolateral intertransverse arthrodesis at the L5-L6 level using 2.0 g autograft per side. The animals were randomly divided into two groups receiving subcutaneous daily injections of either EPO or saline for 20 days. Treatment commenced 2 days preoperatively. Hemoglobin was monitored at baseline and 2, 4, and 6 weeks after fusion surgery. After euthanasia 6 weeks postoperatively, manual palpation, radiographic, and histomorphometric examinations were performed. Bone volume of the fusion mass was estimated by CT after 6 weeks. EPO increased bone fusion volume to 3.85 ccm (3.66-4.05) compared with 3.26 ccm (2.97-3.55) in the control group (p<0.01). EPO treatment improved vascularization of the fusion mass and increased hemoglobin levels (p<0.01). Fusion rate tended to be higher in the EPO group based on manual palpation, CT, and radiographic examinations. For the first time EPO has shown to augment bone formation after autograft PLF in a rabbit model. Increased vascularization provides a partial explanation for the efficacy of EPO as a bone autograft enhancer.     

Tibial epiphyseal development: a cross-sectional histologic and histomorphometric study in the New Zealand white rabbit

Sequential histomorphometric studies on the developing rabbit tibia from birth to skeletal maturity demonstrate that growth plate height lessens as longitudinal growth diminishes. Differing rates of development proximally and distally are documented. Distally, growth plate height, width, and area and total epiphyseal area peak or reach near maximum values by 3 weeks, whereas proximally, they do so by 8 weeks (except for height, which also peaks at 3 weeks). The distal growth plate is being obliterated by 16 weeks, at which time the proximal growth plate remains well structured and open. The distal tibia and fibula develop as one tissue mass. The articular cartilage and epiphyseal cartilage are continuous from birth, whereas a single ossification center and a single growth plate are present by 8 weeks. The data point to the presence of intrinsic growth plate, as well as systemic, control mechanisms affecting skeletal growth. Knowledge of temporal and quantitative features of epiphyseal and growth plate development will greatly aid in the elucidation of the underlying controls.     

The effect of noggin interference in a rabbit posterolateral spinal fusion model

Study design

Noggin protein levels and spinal fusion rates were compared in a rabbit model after application of siRNA against BMP antagonist noggin in paraspinal muscle.

Objective

To test whether endogenous BMPs are sufficient to form bone in the absence of their antagonists, using noggin siRNA to interrupt the negative feedback loop on endogenous BMP within the paraspinal muscles in rabbits.

Summary of background data

Unused Posterolateral lumbar fusion is a standard surgical treatment for many spinal disorders, yet even under ideal conditions the rate of non-fusion approaches 25 %. BMPs are effective in promoting bone formation, and are inhibited by antagonists such as noggin. We have previously shown that in this model, endogenous BMPs are present and endogenous BMP antagonist noggin is strongly increased during spinal fusion. Previous studies have found that noggin siRNA enhanced spinal fusion in combination with supra-physiological amounts of exogenous BMP; however, the effect of the siRNA alone remains unknown.

Methods

A posterolateral intertransverse rabbit lumbar fusion was utilized, as established by Boden et al. SiRNA against noggin was electroporated into paraspinal muscle to determine its effect on fusion. Outcome measures included noggin protein expression, and assessment of spinal fusion at 6 weeks.

Results

SiRNAs were effective in reducing overexpressed noggin in vitro. Noggin protein was successfully knocked down in vivo for the initial 7 days in our rabbit model and returned to detectable levels by 4 weeks and to normal levels by 6 weeks. The overall fusion rate was not significantly enhanced compared to established controls from our earlier work (Tang et al.).

Conclusions

Early noggin suppression does not appear to enhance the BMP activity sufficiently to significantly affect final fusion rates in our model.     

Structural stages in the development of the long bones and epiphyses: a study in the New Zealand white rabbit.

BACKGROUND: Histologic delineation of the events involved in the development of long bones and the developmental age at which these events occur is needed to elucidate the genetic and molecular mechanisms associated with these events. This report describes the sequence of histologic events involved in the formation of long bones and their epiphyses in the New Zealand White rabbit. METHODS: Prenatal studies were performed on twelve, fourteen, fifteen, sixteen, eighteen, twenty-one, twenty-four, and twenty-seven-day-old rabbit embryos, and postnatal studies were performed on newborn rabbits and on three-to-four-day-old; one, two, four, and six-week-old; and two, three, four, six, and eight-month-old rabbits. Histologic specimens from embryos were embedded in plastic and stained with toluidine blue or safranin O-fast green, and specimens from postnatal rabbits were embedded in paraffin and stained with hematoxylin and eosin or safranin O-fast green. RESULTS: Studies of twelve-day-old embryos demonstrated upper and lower limb buds filled with undifferentiated mesenchymal cells, and studies of fourteen-day-old embryos showed mesenchymal condensation and beginning cartilage formation outlining major long bones. Long-bone and epiphyseal development progressed through sixteen structural stages, and the developmental age at which these stages occurred was determined. These stages included limb-bud formation with uniform distribution of mesenchymal cells and formation of an apical ectodermal ridge (stage 1); mesenchymal condensation (stage 2); cartilage differentiation (stage 3); formation of a primary center of ossification (stage 4a); epiphyseal cartilage vascularization with formation of cartilage canals (stage 7); vascular invasion of the developing secondary ossification center (stage 9); bone formation and marrow cavitation in the secondary ossification center with formation of hematopoietic marrow (stage 10); fullest relative extent of secondary-ossification-center development in epiphyseal cartilage (stage 14); thinning of the physis (stage 15); and resorption of the physis with establishment of continuity between epiphyseal and metaphyseal circulations (stage 16).     

Histologic evaluation of absorbable and non-absorbable barrier coated mesh secured to the peritoneum with fibrin sealant in a New Zealand white rabbit model

Purpose  

The purpose of this study is to evaluate the histologic response to fibrin sealant (FS) as an alternative fixation method for laparoscopic ventral hernia repair.     

Effects of noise exposure and hypercholesterolemia on auditory function in the New Zealand white rabbit

An association between hypercholesterolemia and high-frequency hearing loss has been suggested previously. Most data have been epidemiologic, and only recently have experimental studies appeared supporting this observed association. It is unclear whether this hearing loss is related solely to hypercholesterolemia or if it is a consequence of cholesterol-induced vascular changes. The New Zealand White rabbit was used to study the auditory effects of noise, hypercholesterolemia, and the combination of both. Auditory function was evaluated with far-field monitoring of click-evoked auditory brain stem responses by comparing latency/intensity functions and absolute click-evoked thresholds. Hypercholesterolemia was maintained for only 3 weeks, theoretically eliminating atherosclerotic vascular changes as a mechanism contributing to observed changes. Auditory function was unchanged after 3 weeks of hypercholesterolemia. Also, the changes observed after noise exposure were comparable between the normal and the hypercholesterolemic groups. We conclude that if hypercholesterolemia contributes to high-frequency hearing loss, the pathophysiology is related to cholesterol-induced vascular changes, not solely to hypercholesterolemia.