Rabbit Posterolateral Spinal Fusion Model Research Articles

Bioactive Moldable Click Chemistry Polymer Cement with Nano-Hydroxyapatite and Growth Factor-Enhanced Posterolateral Spinal Fusion in a Rabbit Model.

Spinal injuries or diseases necessitate effective fusion solutions, and common clinical approaches involve autografts, allografts, and various bone matrix products, each with limitations. To address these challenges, we developed an innovative moldable click chemistry polymer cement that can be shaped by hand and self-cross-linked in situ for spinal fusion. This self-cross-linking cement, enabled by the bioorthogonal click reaction, excludes the need for toxic initiators or external energy sources. The bioactivity of the cement was promoted by incorporating nanohydroxyapatite and microspheres loaded with recombinant human bone morphogenetic protein-2 and vascular endothelial growth factor, fostering vascular induction and osteointegration. The release kinetics of growth factors, mechanical properties of the cement, and the ability of the scaffold to support in vitro cell proliferation and differentiation were evaluated. In a rabbit posterolateral spinal fusion model, the moldable cement exhibited remarkable induction of bone regeneration and effective bridging of spine vertebral bodies. This bioactive moldable click polymer cement therefore presents a promising biomaterial for spinal fusion augmentation, offering advantages in safety, ease of application, and enhanced bone regrowth.

Long-term posterolateral spinal fusion in rabbits induced by rhBMP6 applied in autologous blood coagulum with synthetic ceramics

Autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (Osteogrow) is a novel therapeutic solution for bone regeneration. This study is aimed to investigate the long-term outcome of ABGS with synthetic ceramics (Osteogrow-C) in rabbit posterolateral spinal fusion (PLF) model. Osteogrow-C implants were implanted bilaterally between rabbit lumbar transverse processes. We compared the outcome following implantation of ABGS with ceramic particles of different chemical composition (TCP and biphasic ceramics containing both TCP and HA) and size (500–1700 µm and 74–420 µm). Outcome was analyzed after 14 and 27 weeks by microCT, histology, and biomechanical analyses. Successful bilateral spinal fusion was observed in all animals at the end of observation period. Chemical composition of ceramic particles has impact on the PLF outcome via resorption of TCP ceramics, while ceramics containing HA were only partially resorbed. Moreover, persistence of ceramic particles subsequently resulted with an increased bone volume in implants with small particles containing high proportion of HA. ABGS (rhBMP6/ABC) with various synthetic ceramic particles promoted spinal fusion in rabbits. This is the first presentation of BMP-mediated ectopic bone formation in rabbit PLF model with radiological, histological, and biomechanical features over a time course of up to 27 weeks.

Application of biomimetic mineralized collagen bone graft material in rabbits posterolateral spinal fusion

To investigate the bone repair and regeneration ability of biomimetic mineralized collagen bone graft material and autologous bone marrow in rabbit posterolateral spinal fusion model. Twenty-seven 20-week-old male New Zealand white rabbits ［weighing (5.0±0.5) kg］ were used to establish the posterolateral spinal fusion model of L 5 and L 6 segments by stripping the transverse process and exposing cancellous bone with electric burr. The rabbits were randomly divided into 3 groups, 9 in each group. Groups A, B, and C were implanted 1.5 mL autologous iliac bone, 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material, and 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material and autologous bone marrow in each bone defect. At 4, 8, and 12 weeks after operation, the apparent hardness of the bone grafting area was observed by manipulation method, in order to evaluate bone graft fusion effects. Three animals were sacrificed in each group at each time point, the vertebral body specimens were excised and the bone defect repair and fusion were observed by X-ray films, and three-dimensional CT examination was performed to evaluate whether new bone was formed in the body. HE staining was performed at each time point to observe the formation of new bone and the repair and fusion of bone defects. The manipulation test showed that bone graft fusion was not found in all groups at 4 weeks after operation; 3 (50.0%), 2 (33.3%), and 4 (66.7%) of groups A, B, and C reached bone graft fusion at 8 weeks after operation; 5 (83.3%), 4 (66.7%), and 5 (83.3%) of groups A, B, and C reached bone graft fusion at 12 weeks after operation; the fusion rate of group C was similar to that of group A, and all higher than that of group B. X-ray film observation showed that the fusion rate of group C at 8 and 12 weeks after operation was higher than that of group B, similar to group A. Three-dimensional CT observation showed that the effect of bone fusion in group C was better than that in group B, which was close to group A. HE staining observation showed that large area of mature lamellar bone coverage appeared in the bone graft area of groups A, B, and C at 12 weeks after operation, the material was completely degraded, and the marginal boundary of the host bone disappeared and tightly combined. Biomimetic mineralized collagen bone graft material mixed with autologous bone marrow has good osteoinduction and osteogenesis guidance. Compared with biomimetic mineralized collagen bone graft material, it has better and faster osteogenesis effect, which is close to autologous bone transplantation.

Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion: 233. Opioids delay healing of spinal fusion: a rabbit posterolateral lumbar fusion model

BACKGROUND CONTEXT Opioid use is prevalent for management of pre- and postoperative pain in patients undergoing spinal fusion. There is evidence that opioids downregulate osteoblasts in-vitro, and one previous study found that morphine delays the maturation and remodeling of callus in a rat femur fracture model. However, the effect of opioids on healing of spinal fusion has not been investigated before. Isolating the effect of opioid exposure in humans would be limited by the numerous confounding factors that affect fusion healing. Therefore, we have used a well-established rabbit model to study the process of spinal fusion healing that closely mimics humans. PURPOSE To study the effect of systemic opioids on the process of healing of spinal fusion in a rabbit posterolateral spinal fusion model. STUDY DESIGN/SETTING Preclinical animal study. PATIENT SAMPLE A total of 24 adult New Zealand white rabbits. OUTCOME MEASURES Assessment of fusion was done by manual palpation, plain radiographs, micro-computed tomography (microCT), and histology. METHODS A total of 24 adult New Zealand white rabbits were studied in two groups after approval from the Institutional Animal Care and Use Committee (IACUC). The opioid group (n=12) received four-weeks preoperative and six-weeks postoperative transdermal fentanyl. Serum fentanyl levels were measured just before surgery and four-weeks postoperatively to ensure adequate levels. The control group (n=12) received only perioperative pain control as necessary. All animals received a bilateral L5-L6 posterolateral spinal fusion using iliac crest autograft. Animals were euthanized at the six-week postoperative time point for analysis. RESULTS A total of 12 animals in control group and 11 animals in the opioid group were available for analysis at the end of six weeks. The fusion scores on manual palpation, radiographs, and microCT were not statistically different. Three-dimensional microCT morphometry found that the fusion mass in the opioid group had a lower bone volume (p=.09), lower trabecular number (p=.02) and higher trabecular separation (p=.02) as compared to control. Histological analysis found areas of incorporation of autograft, and unincorporated graft fragments in both groups. In the control group, there was remodeling of de-novo woven bone to lamellar organization with incorporation of osteocytes, formation of mature marrow, and relative paucity of hypertrophied osteoblasts lining new bone. Sections from the opioid group showed formation of de-novo woven bone, and hypertrophied osteoblasts seen lining the new bone. There were no sections showing lamellar organization and development of mature marrow elements in the opioid group. Less dense trabeculae on microCT correlated with histological findings of relatively immature fusion mass in the opioid group. CONCLUSIONS Systemic opioids led to an inferior quality fusion mass with delay in maturation and remodeling at six-weeks in this rabbit spinal fusion model. These preliminary results lay foundation for further research to investigate underlying cellular mechanisms, temporal fusion process, and dose-duration relationship of opioids responsible for our findings.

Opioids delay healing of spinal fusion: a rabbit posterolateral lumbar fusion model

Background ContextOpioid use is prevalent in the management of pre- and postoperative pain in patients undergoing spinal fusion. There is evidence that opioids downregulate osteoblasts in vitro, and a previous study found that morphine delays the maturation and remodeling of callus in a rat femur fracture model. However, the effect of opioids on healing of spinal fusion has not been investigated before. Isolating the effect of opioid exposure in humans would be limited by the numerous confounding factors that affect fusion healing. Therefore, we have used a well-established rabbit model to study the process of spinal fusion healing that closely mimics humans. PurposeThe objective of this work was to study the effect of systemic opioids on the process of healing of spinal fusion in a rabbit posterolateral spinal fusion model. Study Design/SettingThis is a preclinical animal study. Materials and MethodsTwenty-four adult New Zealand white rabbits were studied in two groups after approval from the Institutional Animal Care and Use Committee (IACUC). The opioid group (n=12) received 4 weeks' preoperative and 6 weeks' postoperative transdermal fentanyl. Serum fentanyl levels were measured just before surgery and 4 weeks postoperatively to ensure adequate levels. The control group (n=12) received only perioperative pain control as necessary. All animals underwent a bilateral L5–L6 posterolateral spinal fusion using iliac crest autograft. Animals were euthanized at the 6-week postoperative time point, and assessment of fusion was done by manual palpation, plain radiographs, microcomputed tomography (microCT), and histology. ResultsTwelve animals in the control group and 11 animals in the opioid group were available for analysis at the end of 6 weeks. The fusion scores on manual palpation, radiographs, and microCT were not statistically different. Three-dimensional microCT morphometry found that the fusion mass in the opioid group had a lower bone volume (p=.09), a lower trabecular number (p=.02), and a higher trabecular separation (p=.02) compared with the control group. Histologic analysis found areas of incorporation of autograft and unincorporated graft fragments in both groups. In the control group, there was remodeling of de novo woven bone to lamellar organization with incorporation of osteocytes, formation of mature marrow, and relative paucity of hypertrophied osteoblasts lining new bone. Sections from the opioid group showed formation of de novo woven bone, and hypertrophied osteoblasts were seen lining the new bone. There were no sections showing lamellar organization and development of mature marrow elements in the opioid group. Less dense trabeculae on microCT correlated with histologic findings of relatively immature fusion mass in the opioid group. ConclusionsSystemic opioids led to an inferior quality fusion mass with delay in maturation and remodeling at 6 weeks in this rabbit spinal fusion model. These preliminary results lay the foundation for further research to investigate underlying cellular mechanisms, the temporal fusion process, and the dose-duration relationship of opioids responsible for our findings.

Combination of bone marrow mesenchymal stem cells sheet and platelet rich plasma for posterolateral lumbar fusion.

Bone tissue engineering provides a substitute for bone transplantation in spinal fusion. This study examined if combined bone marrow-derived mesenchymal stem cells (BMSCs) sheet with platelet-rich plasma (PRP) could promote bone regeneration in a rabbit posterolateral spinal fusion model. BMSCs was isolated and confirmed by Flow cytometric analysis and immunofluorescence staining. The morphology of BMSCs was examined by Hematoxylin and Eosin staining, scanning and transmission electron microscopy. BMSCs were cultured in induction medium or control medium. The osteogenic ability of BMSCs was investigated by various histochemical staining, immunofluorescence staining and qRT-PCR analysis. The BMSCs/PRP was constructed by encapsulating the PRP block with BMSCs sheet. Twenty-four adult rabbits were randomly divided into four groups based on the implanted biomaterials: BMSCs/PRP, BMSCs, iliac crest autograft, and control group. Manual palpation and digital radiography analysis showed that the fusion rate was 100%, 0, 83.3%, and 0 in these 4 groups, respectively. Formation of continuous bone masses in BMSCs/PRP group was confirmed by computed tomography scanning and 3D-reconstruction. These studies demonstrated that BSMCs/PRP significantly accelerated bone regeneration in the rabbit posterolateral spinal fusion model.

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

Noggin protein levels and spinal fusion rates were compared in a rabbit model after application of siRNA against BMP antagonist noggin in paraspinal muscle. 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. 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. 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. 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.). Early noggin suppression does not appear to enhance the BMP activity sufficiently to significantly affect final fusion rates in our model.

Evaluation of a new formulation of demineralized bone matrix putty in a rabbit posterolateral spinal fusion model

Background contextAlternatives to autologous bone graft (ABG) with osteoconductive, osteoinductive, and osteogenic potential continue to prove elusive. Demineralized bone matrix (DBM) however, with its osteoconductive and osteoinductive potential remains a viable option to ABG in posterolateral spine fusion. PurposeTo compare the efficacy of a new formulation of DBM putty with that of ABG in a rabbit posterolateral spinal fusion model. Study designEfficacy of a new formulation of DBM was studied in an experimental animal posterolateral spinal fusion model. MethodsTwenty-four male New Zealand White rabbits underwent bilateral posterolateral spine arthrodesis of the L5–L6 intertransverse processes, using either ABG (control group, n=12) or DBM (DBM made from rabbit bone) putty (test group, n=12). The animals were killed 12 weeks after surgery and the lumbar spines were excised. Fusion success was evaluated by manual palpation, high resolution X-rays, microcomputed tomography imaging, biomechanical four-point bending tests, and histology. ResultsTwo animals were lost because of anesthetic related issues. Manual palpation to assess fusion success in the explanted lumbar spines showed no statistical significant difference in successful fusion in 81.8% (9/11) of DBM group and 72.7% (8/11) of ABG group (p=.99). Reliability of these assessments was measured between three independent observers and found near perfect agreement (intraclass correlation cofficient: 0.92 and 0.94, respectively). Fusion using high resolution X-rays was solid in 10 of the DBM group and 9 of the ABG group (p=.59). Biomechanical testing showed no significant difference in stiffness between the control and test groups on flexion, extension, and left lateral and right lateral bends, with p values accounting for .79, .42, .75, and .52, respectively. The bone volume/total volume was greater than 85% in the DBM treated fusion masses. Histologic evaluation revealed endochondral ossification in both groups, but the fusion masses were more mature in the DBM group. ConclusionsThe DBM putty achieved comparable fusion rates to ABG in the rabbit posterolateral spinal fusion model.

Effect of a single dose of pamidronate administered at the time of surgery in a rabbit posterolateral spinal fusion model

Spinal fusion is usually performed on patients who receive bisphosphonates (BP); however, limited data on their action on spinal fusion are available. Previous studies in animal models have shown that chronic administrations of BP reduced spinal fusion rates, and only one study has shown that a single dose administration of zolendronic acid increased fusion rate. The objective of the present study was to evaluate if pamidronate (PA), which was previously demonstrated to reduce spinal fusion rate when administered continuously for 8 weeks, would increase the spinal fusion rate if administered in a single dose at the time of surgery in a rabbit model. Thirty-two New Zealand rabbits underwent an L5-L6 posterolateral intertransverse fusion with iliac crest autograft. Animals were randomized to receive either PA 3 mg/kg in a single dose immediately after surgery, or normal saline. Animals were killed 8 weeks after surgery and fusion was determined by manual palpation and radiographic analysis. Fusion healing was obtained in eight rabbits (50%) in the PA group and in four animals (25%) in the control group, p = 0.137. In a rabbit model, a single dose of PA did not decrease lumbar spinal arthrodesis consolidation rates, but it obtained a nonsignificant higher spinal fusion rate.

Parathyroid Hormone (1-34) Augments Spinal Fusion, Fusion Mass Volume, and Fusion Mass Quality in a Rabbit Spinal Fusion Model

The posterolateral rabbit spinal fusion model was used to assess the effect of intermittent parathyroid hormone on spinal fusion outcomes. To test the hypothesis that intermittent parathyroid hormone (PTH) improves spinal fusion outcomes in the rabbit posterolateral spinal fusion model. Spinal fusion is the definitive management for spinal deformity or instability, yet despite current technology, 5% to 40% of lumbar fusions result in pseudarthrosis. Animal studies have demonstrated enhanced fracture healing with the use of PTH, but the effect of PTH on spinal fusion is poorly described. Forty-four male New Zealand white rabbits underwent bilateral posterolateral spine fusion (L5-L6 level). Twenty-two rabbits received daily subcutaneous injections of PTH (1-34) (10 microg/kg) and 22 received an injection of saline fluid. All were killed 6 weeks after surgery. L5-L6 vertebral segments were removed and analyzed with manual bending, faxitron radiography, microCT, and histomorphometry. Manual bending identified fusion in 30% (control) versus 81% (PTH) animals (P < 0.001). A radiographic scoring system ("0" = no bone formation, "5" = full fusion) resulted in an average score of 3.36 (control) versus 4.51 (PTH) (P < 0.001). MicroCT analysis demonstrated a median mass of 3.5 cc (control) (range, 2.25-5.40 cc) versus 6.03 cc (PTH) (range, 4.34-10.58 cc) (P < 0.001). Histology showed a median percentage bone area of 14.3% (control) (n = 12) versus 29.9% (PTH) (n = 15) (P < 0.001). The median percentage cartilage was 2.7% (control) (n = 5) versus 26.6% (PTH) (n = 5) (P < 0.01). Osteoclast quantification revealed median values of 140.5 (control) (n = 6) and 345.0 (PTH) (n = 8) (P < 0.001) respectively, and the percentage of osteoblasts revealed a median value of 31.4% (control) (n = 6) versus 64.4% (PTH) (n = 8) (P < 0.001). Intermittent PTH administration increased posterolateral fusion success in rabbits. Fusion bone mass and histologic determinants were also improved with PTH treatment. PTH has promise for use as an adjunctive agent to improve spinal fusion in clinical medicine.

Effect of Hydroxyapatite porous characteristics on healing outcomes in rabbit posterolateral spinal fusion model

Hydroxyapatite (HA) has been commonly used as a bone graft substitute in various kinds of clinical fields. To improve the healing capability of HA, many studies have been performed to reveal its optimal structural characteristics for better healing outcomes. In spinal reconstruction surgery, non-interconnected porous HAs have already been applied as a bone graft extender in order to avoid autogenous bone harvesting. However, there have been few experimental studies regarding the effects of the structural characteristics of HA in posterolateral lumbar intertransverse process spine fusion (PLF). The aims of this study were to investigate the effect of HA porous characteristics on healing outcomes in a rabbit PLF model in order to elucidate appropriate structural characteristics of HA as a bone graft extender. Thirty-six adult female Japanese White rabbits underwent bilateral intertransverse process fusion at the level of L5-6 without internal fixation. We prepared three types of HA with different porosities: HA with 15% porosity (HA15%), HA with 50% porosity (HA50%), and HA with 85% porosity (HA85%), all of which were clinically available materials. The HA15% and HA50% had few interconnecting pores, whereas the HA85%, which was a recently developed material, had abundant interconnecting pores. All rabbits were randomly divided into the following four groups according to the grafted materials: (1) HA15% + autogenous bone, (2) HA50% + autogenous bone, (3) HA85% + autogenous bone, (4) pure autogenous bone graft. The animals were euthanized at 5 weeks after surgery, and post-mortem analyses including biomechanical testing, radiographical and histological evaluations were performed. There was no statistically significant difference in either fusion rate and/or bending stiffness among the three HA groups. However, in histological and radiological analyses, both bone ingrowth rate and direct bone bonding rate in the HA85% group were significantly higher than those in the HA15% and HA50% groups, despite the similar value of bone volume rate in fusion mass among the three HA groups. In the HA85% group, bone ingrowth was achieved throughout the implanted HAs via interconnecting pores and there was excellent unification between the HA granules and the newly mineralized bone. On the other hand, in the non-interconnected porous HA groups, only a little bone ingrowth could be seen at the peripheral pores of the implanted HA, and its surface was mostly covered with fibrous tissue or empty space. The current study demonstrated that the HA porous characteristics had an effect on the histological outcomes in a rabbit PLF model. We would like to conclude that the interconnected high porous structure seems to be promising for the environment of PLF in the point of producing fusion mass with higher cellular viability. This is because the HA85% is superior in terms of integration with the newly formed bone in fusion mass compared to the non-interconnected porous HAs. However, the porous modifications of HA have little influence on fusion rate and mechanical strength because primary stabilization of the fusion segment is mainly achieved by bridging bone between the adjacent transverse processes outside the implanted materials, rather than the degree of integration between the newly formed bone and the HA granules in PLF.

Spinal fusion with recombinant human growth and differentiation factor-5 combined with a mineralized collagen matrix.

The availability of recombinant osteoinductive growth factors and new osteoconductive matrices offers an alternative to the use of autogenous bone (autograft) for grafting indications. This study evaluates the bone-forming activity of a mineralized collagen matrix combined with recombinant human growth and differentiation factor-5 in a rabbit posterolateral spinal fusion model. The activity of three distinct matrix-growth factor formulations is assessed by radiographic, histologic, and mechanical strength methods. Results show that the radiographic density, histologic quality, and mechanical strength of fusion at 12 weeks post-treatment rank consistently within the treatment groups. Optimal formulations are shown to perform similar to autograft in both the rate and strength of fusion. Fusion rates as high as 80% are observed within specific matrix/growth factor formulations. The average biomechanical strength of treated motion segments in the most efficacious formulation is 82% higher than that obtained with autograft, although this difference is not statistically significant. The fusion mass formed in response to matrix/growth factor formulations is composed of normal trabecular bone with a thin outer cortical plate and modest hematopoietic bone marrow. These results demonstrate that the combination of a mineralized collagen matrix with recombinant human growth and differentiation factor-5 maximizes the inherent conductive and inductive properties of each component, respectively, to provide an effective alternative to autograft for bone grafting procedures.