Posterolateral Fusion Model Research Articles

86. Why are DBMs so variable? Influence of fibers, carrier, and tissue bank

BACKGROUND CONTEXT Demineralized bone matrix (DBM) is widely used in spinal fusion. Most DBMs are particles combined with a carrier to provide putty-like handling. Demineralized bone fibers (DBF) are a formulation where allograft bone is demineralized in long fibers so the resulting DBF product is cohesive on its own. It has been suggested that 100% DBF products with no carrier may offer improved performance over particle-based DBMs with carrier. PURPOSE The objective of this study was to evaluate the variability of in vivo performance of DBFs from five different tissue banks with various differences in composition in a posterolateral fusion model. STUDY DESIGN/SETTING Seven different commercially available DBMs were evaluated from five different tissue banks: Four 100% DBFs, two DBFs with carrier, and one particle-based DBM with carrier. PATIENT SAMPLE Not applicable. OUTCOME MEASURES Not applicable. METHODS Single-level posterolateral fusion was performed in athymic rats. Fusion was assessed bilaterally after 4 weeks by manual palpation, radiograph and microCT (mCT) for bridging bone. Fusion mass maturity was assessed using an mCT grading scale and by histology. Statistical analysis was performed using Fishers Exact Test for categorical data, Kruskal-Wallis Test for non-parametric data, and linear regression for the relationship between fusion rate and % carrier. RESULTS Fusion end point assessments of manual palpation, radiography and mCT were closely concordant for all groups. The mCT fusion rates for the 100% DBF groups varied from 0 to 100%. The DBFs with carrier had mCT fusion rates of 0 to 81%. Within the same tissue bank, DBFs with carrier had slightly lower fusion rates than 100% DBF products, although differences did not reach statistical significance. For the DBM particulate with carrier, the fusion rate was 38%. There was an inverse linear correlation between fusion rate and percentage carrier composition (R2 = 0.99) for the carrier-containing groups. Histology and CT maturity grading revealed substantial differences in the quality of bone formation in the fusion mass, even among groups with similar fusion rates. CONCLUSIONS There were large variations in fusion performance for seven commercially available DBF/DBMs in an established preclinical fusion model. The fusion outcomes in this study suggest a detrimental effect of carrier on fusion outcomes. Among 100% DBF products, there were large variations in performance, suggesting that fiber composition alone does not guarantee outcomes. DBM variability is likely driven by variations in allograft processing conditions at different tissue banks, including differences in donor quality, fiber geometry, demineralization, storage, and sterilization procedures. In the absence of definitive clinical evidence, surgeons should carefully consider the tissue bank source using available data in valid animal models when selecting their DBF or DBM grafts. FDA DEVICE/DRUG STATUS Grafton (Approved for this indication), DBX Putty (Approved for this indication), Optium Putty (Approved for this indication).

197. In vitro and in vivo assessment of bone regeneration after local steroid administration using a rodent posterolateral fusion model

BACKGROUND CONTEXT Local intraoperative steroid administration has been shown to reduce soft tissue swelling and dysphagia after cervical spinal fusion. However, the theoretical negative impact of steroid compounds on bone healing has prevented this practice from becoming routine. PURPOSE This study aims to evaluate the effect of local steroid administration on bone regeneration utilizing an established rodent posterolateral fusion model. STUDY DESIGN/SETTING Preclinical animal model PATIENT SAMPLE Female Sprague-Dawley rats (age 12-16 weeks) OUTCOME MEASURES Radiographs, Fusion Scoring, Micro-Computed Tomography, Histology, Gene Expression Analysis METHODS For in vitro assessment, human bone marrow derived mesenchymal stem cells (hBM-MSCs) were grown in media with INFUSETM and different amounts of steroid: vehicle control (no steroid), therapeutic triamcinolone (0.5 mg/kg), and supratherapeutic triamcinolone (2.5 mg/kg). Gene expression analysis of pro-osteogenic markers (RUNX2, Osterix, ALPL, COL1A1, Osteocalcin, Osteopontin), alkaline phosphatase (ALP) activity assays, and alizarin red staining were used to assess for differences in osteogenic differentiation among the treatment groups. For in vivo assessment, 45 female Sprague-Dawley rats underwent bilateral L4-L5 posterolateral fusion (PLF), receiving subtherapeutic doses of INFUSETM (1.0 mg per animal). Additionally, three groups (n = 15 animals each) received either: no steroid (vehicle control), therapeutic triamcinolone (0.5 mg/kg), or supratherapeutic triamcinolone (2.5 mg/kg), administered locally. Spines were harvested at 8 weeks postoperation. Blinded manual palpation by three independent reviewers was used to determine a fusion score, wherein 0 = no fusion, 1 = unilateral bridging bone, and 2 = bilateral bridging bone. The fusion rate was calculated based on the fusion score, in which a score ≥1 indicates successful fusion. New bone volume was quantified with microCT imaging. Representative samples underwent hematoxylin and eosin staining (H&E) for evaluation of fusion. RESULTS All pro-osteogenic transcripts were significantly elevated in samples treated with INFUSETM. Among INFUSETM groups, significant differences in RUNX2, Osterix, COL1A1, and Osteocalcin expression were noted between the different treatment groups: except for Osteocalcin, expression was significantly increased with therapeutic or supra-therapeutic triamcinolone relative to vehicle controls at one or more of the time points tested. ALP activity also increased with triamcinolone treatment relative to controls after 3 days, 3 week, and 3 weeks in culture, and mineral deposition was comparable between all groups. For in vivo assessment, there were no significant differences in average fusion score (FS), fusion rate (FR), or new bone volume (BV) between the control (FS: 0.62 ± 0.59, FR: 38%, BV: 32.4 ± 22.4 mm3), therapeutic steroid (FS: 1.09 ± 0.81, FR: 57%, BV: 40.1 ± 23.2 mm3), and supratherapeutic steroid (FS: 0.58 ± 0.58, FR: 33%, BV: 25.5 ± 21.8 mm3) groups. CONCLUSIONS Local intraoperative steroid, at both therapeutic and supratherapeutic doses, did not have a significant negative effect on multiple markers of bone regeneration in both in vitro and in vivo models. These data suggest that the benefits of local steroid may outweigh any theoretical risk of poor bone healing. FDA DEVICE/DRUG STATUS rhBMP-2 (INFUSE) [non-human use] (Investigational/Not approved)

73. Sex-based difference in response to recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rat posterolateral fusion model

BACKGROUND CONTEXT Despite the known influence of hormones on bone metabolism and osteogenic differentiation, the few clinical studies evaluating outcomes after spine surgery have come to contradictory conclusions as to a sex-based differential response. This study aimed to quantify and compare the bone regenerative response to rhBMP-2 in males vs females in a preclinical setting of spine fusion. PURPOSE The purpose of this study was to determine whether sex influences fusion success, bone regenerative capacity, and bone quality after rhBMP-2-mediated spine fusion. STUDY DESIGN/SETTING Preclinical animal study. PATIENT SAMPLE Male and female Sprague-Dawley rats, ages 12-16 weeks. OUTCOME MEASURES Radiography, fusion scoring, microCT imaging, biomechanics testing. METHODS Twenty-four male and 24 female Sprague Dawley rats underwent L4-L5 posterolateral spine fusion with bilateral placement of an absorbable collagen sponge loaded with 5 μg of rhBMP-2 each (Infuse™; 10 μg/animal). Fusion was evaluated at 8 weeks postoperatively via blinded manual palpation on 14 rats/treatment group using an established scoring system wherein 0=no fusion, 1=unilateral fusion, 2=bilateral fusion, 3=bilateral fusion adjacent to a unilateral fusion, and 4=two adjacent bilateral fusion. A fusion score of 1 or greater (unilateral, single level fusion) was considered successfully fused. Specimens then underwent microCT imaging to evaluate and quantify new bone formation. Biomechanical testing was performed on the remaining 10 rats/group to quantify segmental flexion-extension range of motion and stiffness. RESULTS Both male and female rats achieved 100% fusion rates (with 1-level unilateral bridging considered fused), as assessed by blinded manual palpation. However, fusion scores were greater in male rats when compared with females (female: 2.4 vs males 3.1; p CONCLUSIONS Although a smaller amount of new bone was formed in females treated with Infuse™ relative to their male counterparts, that new bone was denser, with twice the number of trabeculae. Interestingly, female spines exhibited on average a greater segmental flexion-extension range-of-motion, suggesting that biomechanical stabilization may be more dependent upon fusion mass volume than density of the newly formed bone. These results are strongly suggestive of sex-dependent differences in the bone regenerative response to rhBMP-2, and they underscore the need for further study of this phenomenon and the underlying mechanisms involved. FDA DEVICE/DRUG STATUS rhBMP-2: Investigational.

A Comparative Evaluation of Commercially Available Cell-Based Allografts in a Rat Spinal Fusion Model.

To evaluate the comparative abilities of commercially available, viable, cellular bone allografts to promote posterolateral spinal fusion. Human allografts containing live cells were implanted in the athymic rat model of posterolateral spine fusion. Three commercially available allogeneic cellular bone matrices (Trinity Evolution, Trinity ELITE and Osteocel Plus) were compared with syngeneic iliac crest bone as the control. All spines underwent radiographs, manual palpation, and micro-computed tomography (CT) analysis after excision at 6 weeks. Histological sections of randomly selected spines were subjected to semiquantitative histopathological scoring for bone formation. By manual palpation, posterolateral fusion was detected in 40% (6/15) of spines implanted with syngeneic bone, whereas spines implanted with Trinity Evolution and Trinity ELITE allografts yielded 71% (10/14) and 77% (10/13) fusion, respectively. Only 7% (1/14) of spines implanted with Osteocel Plus allografts were judged fused by manual palpation (statistically significantly less than ELITE, P < .0007, and Evolution, P < .0013). The mineralized cancellous bone component of the allografts confounded radiographic analysis, but Trinity Evolution (0.452 ± 0.064) and Trinity ELITE (0.536 ± 0.109) allografts produced statistically significantly higher bone fusion mass volumes measured by quantitative micro-CT than did syngeneic bone (0.292 ± 0.109, P < .0001 for ELITE and P < .003 for Evolution) and Osteocel Plus (0.258 ± 0.103, P < .0001). Semiquantitative histopathological scores supported these findings because the total bone and bone marrow scores reflected significantly better new bone and marrow formation in the Trinity groups than in the Osteocel Plus group. The Trinity Evolution and Trinity ELITE cellular bone allografts were more effective at creating posterolateral fusion than either the Osteocel Plus allografts or syngeneic bone in this animal model. The superior fusion rate of Trinity cellular bone allografts may lead to better clinical outcome of spinal fusion surgeries.

In-vivo Performance of Seven Commercially Available Demineralized Bone Matrix Fiber and Putty Products in a Rat Posterolateral Fusion Model.

Introduction: Demineralized bone matrix (DBM) is a widely used bone graft in spinal fusion. Most commercial DBMs are composed of demineralized bone particles (~125–800 microns) suspended in a carrier that provides improved handling but dilutes the osteoinductive component. DBM fibers (DBF) provide improved osteoconductivity and do not require a carrier. It has been suggested that 100% DBF may offer improved performance over particulate-based DBMs with carrier.Study Design: Seven commercially available DBM products were tested in an athymic rat posterolateral fusion model. There were four 100% DBFs, two DBFs containing a carrier, and one particulate-based DBM containing carrier.Objective: The study objectives were to evaluate the in vivo performance: (1) compare fusion rate and fusion maturity of six commercially available DBFs and one particulate-based DBM, and (2) assess the effect of carrier on fusion outcomes for DBFs in a posterolateral fusion model.Methods: The DBF/DBM products evaluated were: StrandTM Family, Propel® DBM Fibers, Vesuvius® Demineralized Fibers, Optium® DBM Putty, Grafton® DBF, Grafton Flex, and DBX® Putty. Single-level posterolateral fusion was performed in 69 athymic rats. Fusion was assessed bilaterally after 4 weeks by manual palpation, radiograph and CT for bridging bone. Fusion mass maturity was assessed with a CT maturity grading scale and by histology. Statistical analysis was performed using Fishers Exact Test for categorical data and Kruskal-Wallis Test for non-parametric data.Results: Strand Family achieved 100% fusion (18/18) by manual palpation, radiographic and CT evaluation, significantly higher than Propel Fibers, Vesuvius Fibers, Optium Putty, and DBX Putty, and not statistically higher than Grafton DBF and Grafton Flex. Strand Family provided the highest fusion maturity, with CT maturity grade of 2.3/3.0 and 89% mature fusion rate. Fusion results suggest a detrimental effect of carrier on fusion performance.Conclusions: There were large variations in fusion performance for seven commercially available DBM products in an established preclinical fusion model. There were even significant differences between different 100% DBF products, suggesting that composition alone does not guarantee in vivo performance. In the absence of definitive clinical evidence, surgeons should carefully consider available data in valid animal models when selecting demineralized allograft options.

Comparing cellular bone matrices for posterolateral spinal fusion in a rat model.

IntroductionCellular bone matrices (CBM) are allograft products that provide three components essential to new bone formation: an osteoconductive scaffold, extracellular growth factors for cell proliferation and differentiation, and viable cells with osteogenic potential. This is an emerging technology being applied to augment spinal fusion procedures as an alternative to autografts.MethodsWe aim to compare the ability of six commercially‐available human CBMs (Trinity ELITE®, ViviGen®, Cellentra®, Osteocel® Pro, Bio4® and Map3®) to form a stable spinal fusion using an athymic rat model of posterolateral fusion. Iliac crest bone from syngeneic rats was used as a control to approximate the human gold standard. The allografts were implanted at L4‐5 according to vendor specifications in male athymic rats, with 15 rats in each group. MicroCT scans were performed at 48 hours and 6 weeks post‐implantation. The rats were euthanized 6 weeks after surgery and the lumbar spines were harvested for X‐ray, manual palpation and histology analysis by blinded reviewers.ResultsBy manual palpation, five of 15 rats of the syngeneic bone group were fused at 6 weeks. While Trinity ELITE had eight of 15 and Cellentra 11 of 15 rats with stable fusion, only 2 of 15 of ViviGen‐implanted spines were fused and zero of 15 of the Osteocel Pro, Bio4 and Map3 produced stable fusion. MicroCT analysis indicated that total bone volume increased from day 0 to week 6 for all groups except syngeneic bone group. Trinity ELITE (65%) and Cellentra (73%) had significantly greater bone volume increases over all other implants, which was consistent with the histological analysis.ConclusionTrinity ELITE and Cellentra were significantly better than other implants at forming new bone and achieving spinal fusion in this rat model at week 6. These results suggest that there may be large differences in the ability of different CBMs to elicit a successful fusion in the posterolateral spine.

Tissue-engineered bone used in a rabbit model of lumbar intertransverse process fusion: A comparison of osteogenic capacity between two different stem cells.

Spinal fusion serves an important role in the reconstruction of spinal stability via restoration of the normal spinal sequence and relief of pain. Studies have demonstrated that the fusion rate is mainly associated with the osteogenic capacity of the implanted graft. Mesenchymal stem cells (MSCs) have been successfully isolated from human degenerated cartilage endplate (CEP) and designated as CEP-derived stem cells (CESCs). Previous studies have suggested that CESCs possesses in vitro and in vivo chondrogenic potential superior to that of bone marrow (BM)-MSCs. In addition, CESCs have shown a stronger in vitro osteogenic ability. The present study aimed to further determine the in vivo three-dimensional osteogenesis efficacy of CESCs for spinal fusion. Tissue-engineered bone grafts were transplanted into a rabbit model of posterolateral lumbar intertransverse process fusion using CESCs and BM-MSCs as seed cells composited with porous hydroxyapatite (PHA). The results of manual palpation and computed tomography (CT) scan reconstruction indicated that the CESCs/PHA group had a higher fusion rate than the BM-MSCs/PHA group, although the difference was not observed to be statistically significant. In addition, RT-qPCR results revealed that the in vitro CESCs/PHA composite expressed significantly higher levels of osteogenic-specific mRNA compared with the BM-MSCs/PHA composite. Finally, micro-CT and semi-quantitative histological analysis further demonstrated that the newly formed bone quality of the CESCs/PHA group was significantly higher than that of the BM-MSCs/PHA group in the intertransverse process fusion model. Therefore, the study indicated that CESCs possess superior in vivo osteogenesis capacity compared with BM-MSCs, and might serve as an important alternative seed cell source for bone tissue engineering. These results may provide the foundation for a biological solution to spinal fusion or other bone defect issues.

MagnetOs, Vitoss, and Novabone in a Multi-endpoint Study of Posterolateral Fusion: A True Fusion or Not?

Study Design:This study was a multi-endpoint analysis of bone graft substitutes implanted as a standalone graft in a clinically relevant Ovine model of instrumented posterolateral spinal fusion (PLF).Objective:The objective of this study was to obtain high-quality evidence on the efficacy of commercial bone graft substitutes compared with autograft in instrumented PLF using a state-of-the-art model with a complete range of assessment techniques.Summary of Background Data:Preclinical and clinical data on the quality of spinal fusions obtained with bone graft substitutes are often limited. Calcium phosphates with submicron topography have shown promising results in PLF, as these are able to induce bone formation in tissues distant from the host bone, which facilitates bony union.Methods:Nine female, skeletally mature sheep (4–5 y) underwent posterior pedicle screw/rods instrumented PLF at L2–L3 and L4–L5 using the following bone graft materials as a standalone graft per spinal segment: (1) biphasic calcium phosphate with submicron topography (BCP<µm), (2) 45S5 Bioglass (BG), and (3) collagen-β-tricalcium phosphate with a 45S5 Bioglass adjunct (TCP/BG). Autograft bone (AB) was used as a positive control treatment. Twelve weeks after implantation, the spinal segments were evaluated by fusion assessment (manual palpation, x-ray, micro-computed tomography, and histology), fusion mass volume quantification (micro-computed tomography), range of motion (ROM) testing, histologic evaluation, and histomorphometry.Results:Fusion assessment revealed equivalence between AB and BCP<µm by all fusion assessment methods, whereas BG and TCP/BG led to significantly inferior results. Fusion mass volume was highest for BCP<µm, followed by AB, BG, and TCP/BG. ROM testing determined equivalence for spinal levels treated with AB and BCP<µm, while BG and TCP/BG exhibited higher ROM. Histologic evaluation revealed substantial bone formation in the intertransverse regions for AB and BCP<µm, whereas BG and TCP/BG grafts contained fibrous tissue and minimal bone formation. Histologic observations were supported by the histomorphometry data.Conclusions:This study reveals clear differences in efficacy between commercially available bone graft substitutes, emphasizing the importance of clinically relevant animal models with multiendpoint analyses for the evaluation of bone graft materials. The results corroborate the efficacy of calcium phosphate with submicron topography, as this was the only material that showed equivalent performance to autograft in achieving spinal fusion.

Freeze-Dried Platelet-Rich Plasma Induces Osteoblast Proliferation via Platelet-Derived Growth Factor Receptor-Mediated Signal Transduction.

Study DesignControlled laboratory study.Purpose This study aimed to evaluate the in vitro pharmacological activity of growth factors (GFs) in freeze-dried platelet-rich plasma (FD-PRP) after storage for 4 weeks.Overview of LiteratureFreshly prepared PRP is a rich source of many GFs. We reported that FD-PRP stored for 8 weeks accelerated bone union in a rat posterolateral fusion model equally well as fresh-PRP. However, the pharmacological activity of FD-PRP after long-term storage has not been shown in vitro.MethodsImmediately after preparation, as well as 4 weeks after freeze-dried storage, the platelet count was measured. Human osteoblasts were treated with fresh-PRP and FD-PRP, respectively. Western blotting was used to assess the phosphorylation of the platelet-derived growth factor (PDGF) receptor (PDGFR) and its downstream target, extracellular signal-regulated kinase (ERK). The proliferation rates of osteoblasts were investigated by immunocytochemistry and MTT cell viability assays. Furthermore, we used western blotting to evaluate the effect of PDGFR knockdown on the phosphorylation of ERK stimulated with fresh-PRP and FD-PRP.Results Platelet counts in both the fresh-PRP and FD-PRP samples were approximately 10-fold higher than in peripheral blood samples. The phosphorylation and activation of the PDGFR and ERK were evenly induced by fresh-PRP and FD-PRP stimulation. Both freshPRP and FD-PRP significantly induced osteoblast proliferation in MTT cell viability assays. Furthermore, osteoblast PDGFR knockdown attenuated the downstream ERK activation by fresh PRP and FD-PRP.ConclusionsWe demonstrated the pharmacological activity of PDGF in FD-PRP in vitro after 4 weeks of storage.

28. Evaluation of three commercially available synthetic bone grafts in a clinically relevant ovine model of instrumented lumbar posterolateral fusion

BACKGROUND CONTEXT Synthetic bone grafts are important tools for spinal surgeons who wish to avoid the invasive harvesting of autologous bone graft for spinal fusions. Various synthetic bone grafts are available on the market, including materials based on bioglass, calcium phosphate or a combination of both. Side-by-side comparisons of synthetic bone grafts in relevant preclinical study models can provide valuable insights that can aid in the selection of treatment options. PURPOSE The current study compared three commercially available synthetic bone grafts as stand-alone grafts in a clinically relevant ovine model of instrumented posterolateral fusion (PLF). STUDY DESIGN/SETTING The three different synthetic bone grafts evaluated are: (1) putty formulation of biphasic calcium phosphate with submicron topography that was previously shown to have equivalent performance to autograft in this model (BCP; MagnetOs Putty, Kuros Biosciences BV), (2) putty formulation of 45S5 bioglass (BG; Novabone Putty, Novabone Products LLC), (3) composite of beta-tri-calcium phosphate, 45S5 bioglass and bovine type I collagen (TCP+BG; Vitoss BA2X Foam pack, Orthovita, Inc). Autograft bone (AB) was used as positive control. Twelve weeks after implantation, the treatments were evaluated. OUTCOME MEASURES Main study end points include fusion assessment by manual palpation, X-ray imaging and micro-CT scans. Range of motion (ROM) of spine levels was assessed in lateral bending (LB) and flexion-extension (FE). Quantification of fusion mass volume was performed using micro-CT. Histology was performed to evaluate bone formation and tissue responses with the different graft materials. METHODS Following ethical approval, 9 sheep underwent instrumented PLF surgery at L2-3 and L4-5 levels. Autograft was harvested from the Os Ilium and distal femur. After decortication of the transverse processes (TPs), 10 cc of each graft material (AB; n=6, BCP; n=6, BG; n=3, TCP+BG; n=3) was implanted bilaterally in the posterolateral regions, bridging the inter-transverse process space. After sacrifice, specimens were assessed by manual palpation, X-ray imaging, Micro-CT and ROM testing at 7.5 Nm after removing instrumentation. Subsequently, treated spine levels were processed for histology in polymethylmethacrylate to visualize bone tissue in the fusion mass. RESULTS Fusion assessment by manual palpation revealed a 6/6 fusion rate for AB and BCP, while for both BG and TCP+BG, a 1/3 fusion rate was determined. Fusion assessment of X-ray and Micro-CT were consistent and determined an either unilateral or bilateral robust bone bridge in 6/6 levels for both AB and BCP, while The BG group exhibited unilateral bridging in 2/3 levels and the TCP+BG group did not include a robust bone bridge in any of the levels. ROM testing revealed equivalent ROMs in LB and FE for AB and BCP, being substantially and significantly lower than those of BG and TCP+BG groups. Fusion mass volume quantification determined fusion mass volumes of 5.7 ± 1.6 cm3 for AB, 9.6 ± 0.4 cm3 for BCP, 3.0 ± 1.1 cm3 for BG and 2.4 ± 1.0 cm3 for TCP+BG. Correspondingly, histology revealed significant graft resorption for BG and TCP+BG groups, while abundant bone tissue in the inter-TP space was observed for AB and BCP. A continuous bony bridge in between TPs was frequently observed in the BCP group on histological sections. CONCLUSIONS Calcium phosphate with submicron topography was the only synthetic bone graft to perform equally well as autograft as a stand-alone graft in this PLF model. Both bioglass-based bone grafts significantly underperformed as compared to autograft and BCP on every single assessment method. The bioglass-based synthetics were observed to undergo significant resorption and were not able to induce bone formation in the soft tissue region in between the TPs. Calcium phosphate with submicron topography may be the better treatment option than bioglass-based bone grafts as a synthetic bone graft substitute. FDA DEVICE/DRUG STATUS MagnetOs Granules (Investigational/Not approved), Novabone Putty (Approved for this indication), Vitoss BA2X (Approved for this indication).

P9. Dimethyloxaloylglycine (DMOG) preconditioning of adipose-derived mesenchymal stem cells as strategy for enhancing posterolateral lumbar fusion in a rat model

BACKGROUND CONTEXT Transplantation of adipose-derived mesenchymal stem cells (ADSCs) has become a promising approach for bone regeneration therapies, particularly in elderly patients. However, poor cell survival in vivo after implantation due to the ischemic tissue environment has been one of the major limitations of the effectiveness of this strategy. Hypoxia Inducible Factor-1α (HIF-1α) plays an important role in angiogenic-osteogenic coupling during bone regeneration, as it is a critical mediator of the adaptive cell response to hypoxia. DMOG treatment has been demonstrated to stabilize cellular expression of HIF-1α in cells, as well as enhance osteogenic and angiogenic capacity of MSCs. By therapeutically targeting both osteogenesis and vascularization via DMOG preconditioned MSCs, this tissue-engineering approach sought to yield superior bone quality and integration, thereby increasing rates of spinal fusion. PURPOSE We aimed to demonstrate that preconditioning adipose (ADSC)-derived (AD) MSCs with DMOG prior to implantation will prolong survival in vivo and improve spinal fusion outcomes in a rat model. STUDY DESIGN/SETTING In vivo study via a rat posterolateral spinal fusion model. PATIENT SAMPLE Syngeneic 6-8 week old Lewis rats. OUTCOME MEASURES Manual palpation scoring was performed by blinded researchers as follows: 0 = non-fused; 1 = partial fusion, some motion across operative joint; and, 2 = fused, no motion across the operated joint. MicroCT images were used to assess fusion mass volume (mm^3) via ImageJ software and determine CT fusion score (0 = non-fused; 1 = unilateral fusion; 2 = bilateral fusion). METHODS ADSCs were isolated from the inguinal fat pads of syngeneic 6-8 week old Lewis rats and cultured in vitro. When passage 1 (P1) ADSCs reached approximately 80% confluency they were pre-conditioned for 24 hours with 1 ng of DMOG (Fisher Scientific). After pre-conditioning, 2 × 10^6 ADSCs were seeded onto Vitoss (Stryker) bone graft substitute scaffolds for subsequent transplantation. Posterolateral spinal fusion surgery at L4-L5 was performed on 18 female Lewis rats divided into 2 experimental groups: [1] Vitoss+ADSCs pre-conditioned with DMOG; and, [2] Vitoss+non pre-conditioned ADSCs. Fusion was evaluated eight weeks post-surgery. Manual palpation scoring evaluated the motion across the operated joint, and MicroCT images were assessed for fusion mass volume and CT fusion. RESULTS Preliminary microCT imaging data suggest that DMOG pre-conditioned ADSCs (n=12) yielded significantly higher fusion mass volumes than non-preconditioned ADSCs (n=6) (23.49 mm^3 vs. 15.39 mm^3, respectively, p=.001). DMOG pre-conditioned ADSCs showed a trend towards higher rates of fusion and manual palpation scores compared to non-conditioned ADSCs (1.16 vs. 0.50, respectively, p=.06; and 1.25 vs. 0.66, respectively, p>.05). CONCLUSIONS In our rat posterolateral fusion model, DMOG pre-conditioned ADSCs displayed an increased fusion mass volume, CT fusion rates, and manual palpation score compared to non-conditioned ADSCs. Ongoing histological studies will evaluate whether there are any differences in the quality of bone formed within the fusion masses. Future studies will also compare whether these results are also similar in bone marrow-derived MSCs. FDA DEVICE/DRUG STATUS Dimethyloxaloylglycine (Investigational/Not approved)

Effect of local zoledronic acid administration in a rat model of posterolateral spinal fusion

Study designRandomized animal model study. PurposePosterolateral spinal fusion represents a common surgical procedure in the United States. The effect of bisphosphonate administration in these patients is poorly understood. The purpose of this study is to determine whether local administration of bisphosphonate by soaking bone autograft would affect the apparent bone density or structural properties of the fusion mass in a rat model of posterolateral spinal fusion. Methods36 Spring Dawley rats underwent L4-5 posterolateral spinal fusion with bone autograft. These rats were divided into three groups, two experimental groups and one control group. Each of the experimental groups underwent spinal fusion with morselized vertebral cortical and cancellous autograft soaked in zoledronic acid solution; one group 20 mcg/mL, another 200 mcg/mL. The control group underwent L4-5 spinal fusion with cancellous allograft soaked with saline. At 8 weeks, the rats were euthanized for analysis. Evaluations consisted of micro-CT scanning, four-point bending biomechanical testing, histology, and radiographs. ResultsBoth of the experimental groups showed statistically significant increase in apparent bone density and bone volume fraction at the fusion mass. Biomechanical measures revealed a trend for improvement in the experimental groups, but these did not reach statistical significance. ConclusionsThis data suggest that locally administered bisphosphonate medications result in increased apparent bone density and bone volume fraction at the fusion mass in posterolateral spinal fusion, and that there appear to be no deleterious consequences with regards to the stiffness or maximum load to failure of the fusion mass under flexion bending evaluation.

Positive effect on spinal fusion by the combination of platelet-rich plasma and collagen-mineral scaffold using lumbar posterolateral fusion model in rats

BackgroundPlatelet-rich plasma (PRP) is autologous in origin and contains a high concentration of platelets which is a source of various growth factors. Previous studies have suggested that PRP has a positive effect in accelerating fusion by an autologous bone graft in a lumbar fusion. The role of PRP on artificial bone grafts in spinal fusion remains controversial. In this study, positive effect on spinal fusion by PRP was hypothesized; in vitro and in vivo studies were designed to test this hypothesis.MethodsPRP was produced from peripheral blood of Sprague-Dawley (SD) rats. A lumbar posterolateral arthrodesis model was used to test the efficacy of PRP on spinal fusion. Thirty SD rats were divided into three groups by different implants: the PRP group, PRP plus collagen-mineral carrier; the platelet-poor plasma (PPP) group, PPP plus collagen-mineral carrier; and the control group, collagen-mineral only. Spinal fusion was examined using plain radiographs, micro-computed tomography (micro-CT), manual palpation, and histological analysis. The fusion rate by micro-CT and that by manual palpation in groups were compared.ResultsIn the micro-CT results, 16 fused segments were observed in the PRP group (80%, 16/20), 2 in the PPP group (10%, 2/20), and 2 in the control group (10%, 2/20). The fusion rate, determined by manual palpation, was 60% (6/10) in the PRP group, 0% (0/10) in the PPP group, and 0% (0/10) in the control group. Histology showed that the PRP group had more new bone and matured marrow formation.ConclusionsThe results of this study demonstrated that PRP on an artificial bone carrier had positive effects on lumbar spinal fusion in rats. In the future, this composite could be potentially used as a bone graft in humans.

Biphasic calcium phosphate with submicron surface topography in an Ovine model of instrumented posterolateral spinal fusion.

As spinal fusions require large volumes of bone graft, different bone graft substitutes are being investigated as alternatives. A subclass of calcium phosphate materials with submicron surface topography has been shown to be a highly effective bone graft substitute. In this work, a commercially available biphasic calcium phosphate (BCP) with submicron surface topography (MagnetOs; Kuros Biosciences BV) was evaluated in an Ovine model of instrumented posterolateral fusion. The material was implanted stand‐alone, either as granules (BCPgranules) or as granules embedded within a fast‐resorbing polymeric carrier (BCPputty) and compared to autograft bone (AG). Twenty‐five adult, female Merino sheep underwent posterolateral fusion at L2‐3 and L4‐5 levels with instrumentation. After 6, 12, and 26 weeks, outcomes were evaluated by manual palpation, range of motion (ROM) testing, micro‐computed tomography, histology and histomorphometry. Fusion assessment by manual palpation 12 weeks after implantation revealed 100% fusion rates in all treatment groups. The three treatment groups showed a significant decrease in lateral bending at the fusion levels at 12 weeks (P < 0.05) and 26 weeks (P < 0.001) compared to the 6 week time‐point. Flexion‐extension and axial rotation were also reduced over time, but statistical significance was only reached in flexion‐extension for AG and BCPputty between the 6 and 26 week time‐points (P < 0.05). No significant differences in ROM were observed between the treatment groups at any of the time‐points investigated. Histological assessment at 12 weeks showed fusion rates of 75%, 92%, and 83% for AG, BCPgranules and BCPputty, respectively. The fusion rates were further increased 26 weeks postimplantation. Similar trends of bone growth were observed by histomorphometry. The fusion mass consisted of at least 55% bone for all treatment groups 26 weeks after implantation. These results suggest that this BCP with submicron surface topography, in granules or putty form, is a promising alternative to autograft for spinal fusion.

Feasibility and effects of a self-assembling peptide as a scaffold in bone healing: An in vivo study in rabbit lumbar posterolateral fusion and tibial intramedullary models.

Spinal fusion and bone defects after injuries, removal of bone tumors, and infections require repair by implantation. In this study, we show self-assembling peptide (SPG-178) hydrogel-induced bone healing in vivo. Posterolateral lumbar fusion and tibial intramedullary models of rabbits were prepared. In the tibia model, micro-CT analysis revealed a significantly higher degree of newly formed bone matrix in the SPG-178 group compared to the other groups. SEM/3D micrographs showed that the cavity filled with SPG-178 had collagen fibers attached to host bone. After 28 days, samples from the SPG-178 group showed significant repair of the defect. In the posterolateral lumbar fusion models, micro-CT showed a tendency for a higher degree of newly formed bone matrix in the SPG-178 group compared to the β-TCP and bone chips only groups. Von Kossa staining showed marked new bone formation attached to the lamina that was most prominent at the implanted SPG-178 composite margin. SPG-178 is a material that is likely to be used in clinical applications because it has several benefits. These include its favorable bone conduction properties, its ability to act as a support for various cells and growth factors, its lack of infection risk compared with materials of animal origin such as ECM, and the ease with which it can be used to fill defects with complex shapes and be combined with a wide range of other materials. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3285-3293, 2018.

Thursday, September 27, 2018 2:05 PM–3:05 PM Section on Biologics and Basic Research Abstract Presentations: 128. Comparative study of cellular bone matrices for posterolateral spinal fusion

BACKGROUND CONTEXT Cellular bone matrices (CBM) are allograft products that combine demineralized bone with viable cancellous bone prepared to retain cells with skeletal tissue differentiation capabilities. Using an athymic rat model of posterolateral fusion, several commercially-available human CBMs were compared: Trinity ELITE (TEL; OrthoFix, Lewisville, TX, United States), ViviGen (VIV; DePuy Synthes, Raynham, MA, United States), Cellentra (CEL; Zimmer Biomet, Warsaw, IN, United States), Osteocel Pro (OCP; NuVasive, San Diego, CA, United States), Bio4 (BIO; Stryker, Kalamazoo, MI, United States) and map3 (MAP; RTI Surgical, Marquette, MI, United States). Iliac crest bone from syngeneic rats was used as a control to approximate the human gold standard. METHODS To allow for lot-to-lot variability, three different lots of each implant type were procured and each was used to implant five rats, for a total of 15 9-10 week old male rats per implant type. Each implant was stored, thawed, and prepared according to the manufacturer's instructions and all implantations occurred within the manufacturers’ time allowance for use after preparation. After anesthesia and preparation, a single posterior midline longitudinal skin and subcutaneous incision and bilateral longitudinal paraspinal myofascial incisions were made to expose the transverse processes and intertransverse membranes at the L4-5 level. The processes were decorticated and 0.3 cc of allograft implant or freshly harvested syngeneic iliac bone graft was placed bilaterally. Surgeons were blinded as to which implant was which. Incisions were closed with sutures to avoid any interference with in vivo animal microCT scans performed within 48 hours of surgery on all rats. The rats were euthanized 6 weeks after surgery and the lumbar spines harvested. A second microCT scan was taken and compared with the microCT taken at the time of implantation to assess fusion and bone remodeling. Stable fusion was also evaluated by manual palpation by three independent, blinded reviewers. MicroCT analysis was performed by an independent CRO (ImageIQ, Cleveland, OH, USA). All assessments were done by experienced evaluators blinded to the treatments. Anonymity of implant type was rigorously kept to avoid any bias. RESULTS By manual palpation, 5/15 (33%) rats of the syngeneic bone group were fused at 6 weeks. While TEL had 8/15 (53%) and CEL 11/15 (73%) rats with stable fusion, only 2/15 (13%) of VIV-implanted spines were fused and 0/15 (0%) of the OCP, BIO and MAP CBMs produced stable fusion. The TEL and CEL groups were significantly different from the other CBMs but not different from each other. MicroCT analysis indicated percentage increases in bone volume from the first measurement to the second for all groups, but only TEL and CEL had significant increases over the syngeneic bone control (TEL 65% and CEL 73% vs. syngeneic bone 21%, respectively). CONCLUSIONS Because the CBMs tested have distinct formulations and are likely processed differently, it is not surprising that the results would be different between the groups. While map3 has cells added at the time of surgery, the other CBMs are processed with cells adherent to the bone matrix. The claimed live cell and stem cell contents differ between products. Trinity ELITE and Cellentra were found to be significantly better than other implants at forming new bone and achieving fusion. The other CBMs did not produce greater bone formation than the control and were very poor at forming a solid fusion. These results suggest that there may be large differences in the ability of different CBMs to elicit a successful fusion in the posterolateral spine. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

The Effects of High-Dose Parathyroid Hormone Treatment on Fusion Outcomes in a Rabbit Model of Posterolateral Lumbar Spinal Fusion Alone and in Combination with Bone Morphogenetic Protein 2 Treatment

Parathyroid hormone (PTH) (1-34) treatment reduces fracture risk in osteoporotic patients. Previously, we demonstrated in a rabbit model that low-dose PTH treatment resulted in increased fusion mass volume. As effects of PTH on bone are dose-dependent, we aimed to evaluate whether increasing dosage of PTH increases both volume and biomechanical stiffness of the resulting fusion masses and/or exhibits synergistic effects with low-dose bone morphogenetic protein 2 (BMP-2). Posterolateral lumbar spinal fusion surgery was performed on 60 New Zealand White rabbits divided into 6 experimental groups: iliac crest autograft alone, autograft plus 20 μg/kg/day PTH, autograft plus 40 μg/kg/day PTH, BMP-2 alone, BMP-2 plus 20 μg/kg/day PTH, and BMP-2 plus 40 μg/kg PTH. Fusion was assessed at postoperative week 6 via manual palpation, volumetric computed tomography analysis, and 4-point bending biomechanical testing. All groups treated with BMP-2 fused. Increasing doses of PTH resulted in increased fusion mass volume compared with autograft alone. Autograft plus 40 μg/kg/day PTH yielded fusion mass volumes comparable to BMP-2. When the autograft groups were considered alone, increased mechanical stiffness was observed only in the 20 μg/kg/day group. No significant stiffness differences were observed between BMP-2 groups. Treatment with the highest dose of PTH resulted in fusion mass volumes similar to those obtained with BMP-2. When the autograft groups were considered alone, significant increases in mechanical stiffness were observed at a dosage of 20 μg/kg/day, suggesting there may be an optimal dose of PTH in the rabbit model. Effects of BMP-2 on fusion were dominant.

Effect of modulating dietary vitamin D on the general bone health of rats during posterolateral spinal fusion.

Vitamin D plays a significant role in musculoskeletal health by regulating calcium, phosphate, and promoting new bone mineralization. The purpose of this study was to understand the effect of dietary vitamin D on general bone health during peri-operative bone healing via an in vivo dosing study of vitamin D in a rat posterolateral fusion model using autograft. Vitamin D Deficient (DD), vitamin D Insufficient (ID), Control vitamin D (CD), and Hyper-vitamin D (HD) groups were studied. Increasing dietary vitamin D improved quantitative measures of femoral geometry, including femoral strength, stiffness, and density. Femoral biomechanics, cortical thickness, moment of inertia, cross-sectional area, and measures from bone ashing were all greater in the HD group versus the CD. This suggests that additional dietary vitamin D above normal levels during spinal fusion may lead to improvement in bone health. Serum vitamin D levels were also observed to decrease during fusion healing. These results demonstrate that dietary vitamin D improves general bone health in the femur of a rat model during posterolateral spinal fusion. This suggests a role for further clinical evaluation of vitamin D dietary intake during the peri-operative period, with the possibility of avoiding adverse consequences to general bone health. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1435-1443, 2018.

Effect of Freeze-Dried Allograft Bone With Human Basic Fibroblast Growth Factor Containing a Collagen-Binding Domain From Clostridium histolyticum Collagenase on Bone Formation After Lumbar Posterolateral Fusion Surgery in Rats.

An experimental study. To evaluate the effectiveness of freeze-dried bone allograft (FDBA) with basic fibroblast growth factor (bFGF) fused with the polycystic kidney disease domain (PKD) and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase, for the acceleration of lumbar posterolateral fusion in rats. Reports indicate bFGF is an effective growth factor with osteogenic potential for promoting bone regeneration, although its efficiency decreases rapidly following its diffusion in body fluid from the host site. We developed a bFGF fusion protein containing the PKD and the CBD of C histolyticum collagenase (bFGF-PKD-CBD), which markedly enhanced bone formation at a relatively low concentration when applied to the surface of rat femurs in a previous study. The potential of this novel protein to accelerate bone fusion in a rat model of lumbar posterolateral fusion has yet to be investigated. Bilateral L4-L5 posterolateral fusions were performed, using 150 mg of FDBA powder per side. A total of 20 male Sprague-Dawley rats weighing 200 to 250 g/each were divided into two groups of 10 rats: FDBA was incubated with either phosphate-buffered saline (control group) or 0.58 nmol bFGF-PKD-CBD (bFGF-PKD-CBD group) before fusion surgery. The effect of bFGF-PKD-CBD was estimated using radiographs, microcomputed tomography, and histology (hematoxylin-eosin and von Kossa staining). Both grafted bone volume in the posterolateral lesion and the volume of new bone formation on the surface of laminae and spinal processes were significantly higher in the bFGF-PKD-CBD group than in the control group. Histologically, new bone formation and surrounding chondrocytes and fibroblasts were prominent in the bFGF-PKD-CBD group. FDBA infused with bFGF-PKD-CBD may be a promising material for accelerating spinal fusion, and the FDBA-based delivery system for localizing bFGF-PKD-CBD may offer novel therapeutic approaches to augment spinal fusion. N/A.

Injectable and compression-resistant low-viscosity polymer/ceramic composite carriers for rhBMP-2 in a rabbit model of posterolateral fusion: a pilot study

BackgroundThe challenging biological and mechanical environment of posterolateral fusion (PLF) requires a carrier that spans the transverse processes and resists the compressive forces of the posterior musculature. The less traumatic posterolateral approach enabled by minimally invasive surgical techniques has prompted investigations into alternative rhBMP-2 carriers that are injectable, settable, and compression-resistant. In this pilot study, we investigated injectable low-viscosity (LV) polymer/composite bone grafts as compression-resistant carriers for rhBMP-2 in a single-level rabbit PLF model.MethodsLV grafts were augmented with ceramic microparticles: (1) hydrolytically degradable bioactive glass (BG), or (2) cell-degradable 85% β-tricalcium phosphate/15% hydroxyapatite (CM). Material properties, such as pore size, viscosity, working time, and bulk modulus upon curing, were measured for each LV polymer/ceramic material. An in vivo model of posterolateral fusion in a rabbit was used to assess the grafts’ capability to encourage spinal fusion.ResultsThese materials maintained a working time between 9.6 and 10.3 min, with a final bulk modulus between 1.2 and 3.1 MPa. The LV polymer/composite bone grafts released 55% of their rhBMP-2 over a 14-day period. As assessed by manual palpation in vivo, fusion was achieved in all (n = 3) animals treated with LV/BG or LV/CM carriers incorporating 430 μg rhBMP-2/ml. Images of μCT and histological sections revealed evidence of bone fusion near the transverse processes.ConclusionThis study highlights the potential of LV grafts as injectable and compression-resistant rhBMP-2 carriers for posterolateral spinal fusion.