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1.  Enhancement of Tendon–Bone Healing for Anterior Cruciate Ligament (ACL) Reconstruction Using Bone Marrow-Derived Mesenchymal Stem Cells Infected with BMP-2 
At present, due to the growing attention focused on the issue of tendon–bone healing, we carried out an animal study of the use of genetic intervention combined with cell transplantation for the promotion of this process. Here, the efficacy of bone marrow stromal cells infected with bone morphogenetic protein-2 (BMP-2) on tendon–bone healing was determined. A eukaryotic expression vector containing the BMP-2 gene was constructed and bone marrow-derived mesenchymal stem cells (bMSCs) were infected with a lentivirus. Next, we examined the viability of the infected cells and the mRNA and protein levels of BMP-2-infected bMSCs. Gastrocnemius tendons, gastrocnemius tendons wrapped by bMSCs infected with the control virus (bMSCs+Lv-Control), and gastrocnemius tendons wrapped by bMSCs infected with the recombinant BMP-2 virus (bMSCs+Lv-BMP-2) were used to reconstruct the anterior cruciate ligament (ACL) in New Zealand white rabbits. Specimens from each group were harvested four and eight weeks postoperatively and evaluated using biomechanical and histological methods. The bMSCs were infected with the lentivirus at an efficiency close to 100%. The BMP-2 mRNA and protein levels in bMSCs were significantly increased after lentiviral infection. The bMSCs and BMP-2-infected bMSCs on the gastrocnemius tendon improved the biomechanical properties of the graft in the bone tunnel; specifically, bMSCs infected with BMP-2 had a positive effect on tendon–bone healing. In the four-week and eight-week groups, bMSCs+Lv-BMP-2 group exhibited significantly higher maximum loads of 29.3 ± 7.4 N and 45.5 ± 11.9 N, respectively, compared with the control group (19.9 ± 6.4 N and 21.9 ± 4.9 N) (P = 0.041 and P = 0.001, respectively). In the eight-week groups, the stiffness of the bMSCs+Lv-BMP-2 group (32.5 ± 7.3) was significantly higher than that of the bMSCs+Lv-Control group (22.8 ± 7.4) or control groups (12.4 ± 6.0) (p = 0.036 and 0.001, respectively). Based on the histological findings, there was an increased amount of perpendicular collagen fibers formed between the tendon and bone in the bMSCs+Lv-Control and bMSCs+Lv-BMP-2 group, compared with the gastrocnemius tendons. The proliferation of cartilage-like cells and the formation of fibrocartilage-like tissue were highest within the bone tunnels in the bMSCs+Lv-BMP-2 group. These results suggest that this lentivirus can be used to efficiently infect bMSCs with BMP-2. Furthermore, tendons wrapped by bMSCs+Lv-BMP-2 improved tendon–bone healing.
PMCID: PMC3497344  PMID: 23202970
tendon-bone healing; anterior cruciate ligament (ACL); reconstruction; bone marrow-derived mesenchymal stem cells
2.  Effect of hyperbaric oxygen on mesenchymal stem cells for lumbar fusion in vivo 
Hyperbaric oxygen (HBO) therapy has been proved in improving bone healing, but its effects on mesenchymal stem cells (MSCs) in vivo is not clear. The aims of this study are to clarify whether the HBO therapy has the same enhancing effect on MSCs with regard to bone formation and maturation and to ascertain whether the transplanted MSCs survive in the grafted area and contribute to new bone formation.
Twenty-three adult rabbits underwent posterolateral fusion at L4-L5 level. The animals were divided into three groups according to the material implanted and subsequent treatment: (1) Alginate carrier (n = 6); (2) Alginate-MSCs composite (n = 11); and (3) Alginate-MSCs composite with HBO therapy (n = 6). After 12 weeks, spine fusion was examined using radiographic examination, manual testing, and histological examination. Using a PKH fluorescence labeling system, whether the transplanted MSCs survived and contributed to new bone formation in the grafted area after HBO therapy was also examined.
The bilateral fusion areas in each animal were evaluated independently. By radiographic examination and manual palpation, union for the Alginate, Alginate-MSCs, and Alginate-MSCs-HBO groups was 0 of 12, 10 of 22, and 6 of 12 respectively. The difference between the Alginate-MSCs and Alginate-MSCs-HBO groups was not significant (P = 0.7997). The fluorescence microscopy histological analysis indicated that the transplanted PKH67-labeled MSCs survived and partly contributed to new bone formation in the grafted area.
This study demonstrated that the preconditioned MSCs could survive and yield bone formation in the grafted area. HBO therapy did not enhance the osteogenic ability of MSCs and improve the success of spine fusion in the rabbit model. Although there was no significant effect of HBO therapy on MSCs for spine fusion, the study encourages us to research a more basic approach for determining the optimal oxygen tension and pressure that are required to maintain and enhance the osteogenic ability of preconditioned MSCs. Further controlled in vivo and in vitro studies are required for achieving a better understanding of the effect of HBO treatment on MSCs.
PMCID: PMC2850325  PMID: 20302633
3.  Effect of Hydroxyapatite porous characteristics on healing outcomes in rabbit posterolateral spinal fusion model 
European Spine Journal  2007;16(12):2215-2224.
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.
PMCID: PMC2140139  PMID: 17891422
Bone graft substitute; Hydroxyapatite; Spine fusion; Porous characteristics; Interconnecting pore
4.  Correlative radiological, self-assessment and clinical analysis of evolution in instrumented dorsal and lateral fusion for degenerative lumbar spine disease. Autograft versus coralline hydroxyapatite 
European Spine Journal  2005;14(7):630-638.
This prospective longitudinal randomized clinical and radiological study compared the evolution of instrumented posterolateral lumbar and lumbosacral fusion using either coralline hydroxyapatite (CH), or iliac bone graft (IBG) or both in three comparable groups, A, B and C, which included 19, 18 and 20 patients, respectively, who suffered from symptomatic degenerative lumbar spinal stenosis and underwent decompression and fusion. The patients were divided randomly according to the graft used and the side that it was applied. The spines of group A received autologous IBG bilaterally; group B, IBG on the left side and hydroxyapatite mixed with local bone and bone marrow on the right side; group C, hydroxyapatite mixed with local bone and bone marrow bilaterally. The age of the patients in the groups A, B and C was 61±11 years, 64±8 years and 58±8 years, respectively. The SF-36, Oswestry Disability Index (ODI), and Roland-Morris (R-M) surveys were used for subjective evaluation of the result of the surgery and the Visual Analogue Scale (VAS) for pain severity. Plain roentgenograms including anteroposterior, lateral and oblique views, and lateral plus frontal bending views of the instrumented spine and CT scan were used to evaluate the evolution of the posterolateral fusion in all groups and sides. Two independent senior orthopaedic radiologists were asked to evaluate first the evolution of the dorsolateral bony fusion 3–48 months postoperatively with the Christiansen’s radiologic method, and secondly the hydroxyapatite resorption course in the spines of groups B and C. The diagnosis of solid spinal fusion was definitively confirmed with the addition of the bending views, CT scans and self-assessment scores. The intraobserver and interobserver agreement (r) for radiological fusion was 0.71 and 0.69, respectively, and 0.83 and 0.76 for evaluation of CH resorption. T12−S1 lordosis and segmental angulation did not change postoperatively. There was no radiological evidence for non-union on the plain roentgenograms and CT scans. Radiological fusion was achieved 1 year postoperatively and was observed in all groups and vertebral segments. Six months postoperatively there was an obvious resorption of hydroxyapatite granules at the intertransverse intersegmental spaces in the right side of the spines of group B and both sides of group C. The resorption of hydroxyapatite was completed 1 year postoperatively. Bone bridging started in the third month postoperatively in all instrumented spines and all levels posteriorly as well as between the transverse processes in the spines of the group A and on the left side of the spines of group B where IBG was applied. SF-36, ODI, and R-M score improved postoperatively in a similar way in all groups. There was one pedicle screw breakage at the lowermost instrumented level in group A and two in group C without radiologically visible pseudarthrosis, which were considered as having non-union. Operative time and blood loss were less in the patients of group C, while donor site complaints were observed in the patients of the groups A and B only. This study showed that autologous IBG remains the “gold standard” for achieving solid posterior instrumented lumbar fusion, to which each new graft should be compared. The incorporation of coralline hydroxyapatite mixed with local bone and bone marrow needs adequate bleeding bone surface. Subsequently, hydroxyapatite was proven in this series to not be appropriate for intertransverse posterolateral fusion, because the host bone in this area is little. However, the use of hydroxyapatite over the decorticated laminae that represents a wide host area was followed by solid dorsal fusion within the expected time.
PMCID: PMC3489222  PMID: 15789231
Coralline hydroxyapatite; Lumbar spinal stenosis; Instrumented fusion
5.  An injectable method for noninvasive spine fusion 
The Spine Journal  2011;11(6):545-556.
Bone morphogenetic proteins (BMPs) induce bone formation but are difficult to localize, and subsequent diffusion from the site of interest and short half-life reduce the efficacy of the protein. Currently, spine fusion requires stripping, decortications of the transverse processes, and an autograft harvest procedure. Even in combination with BMPs, clinical spinal fusion has a high failure rate, presumably because of difficulties in localizing sufficient levels of BMP.
The goal was to achieve reliable spine fusion through a single injection of a cell-based gene therapy system without the need for any surgical intervention.
Eighty-seven immunodeficient (n=44) and immune-competent (n=43) mice were injected along the paraspinous musculature to achieve rapid induction of heterotopic ossification (HO) and ultimately spinal arthrodesis.
Immunodeficient and immune-competent mice were injected with fibroblasts, transduced with an adenoviral vector to express BMP2, along the paraspinous musculature. Bone formation was evaluated via radiographs, microcomputed tomography, and biomechanical analysis.
ew bridging bone between the vertebrae and the fusion to adjacent skeletal bone was obtained as early as 2 weeks. Reduction in spine flexion-extension also occurred as early as 2 weeks after injection of the gene therapy system, with greater than 90% fusion by 4 weeks in all animals regardless of their genetic background.
Injection of our cell-based system into the paraspinous musculature induces spinal fusion that is dependent neither on the cell type nor on the immune status. These studies are the first to harness HO in an immune-competent model as a noninvasive injectable system for clinically relevant spinal fusion and may one day impact human spinal arthrodesis.
PMCID: PMC3327508  PMID: 21292563
Gene therapy; Spine fusion; Heterotopic ossification; BMP2; Spinal arthrodesis
6.  Comparison of the effects of bilateral posterior dynamic and rigid fixation devices on the loads in the lumbar spine: a finite element analysis 
European Spine Journal  2007;16(8):1223-1231.
A bilateral dynamic stabilization device is assumed to alter favorable the movement and load transmission of a spinal segment without the intention of fusion of that segment. Little is known about the effect of a posterior dynamic fixation device on the mechanical behavior of the lumbar spine. Muscle forces were disregarded in the few biomechanical studies published. The aim of this study was to determine how the spinal loads are affected by a bilateral posterior dynamic implant compared to a rigid fixator which does not claim to maintain mobility. A paired monosegmental posterior dynamic implant was inserted at level L3/L4 in a validated finite element model of the lumbar spine. Both a healthy and a slightly degenerated disc were assumed at implant level. Distraction of the bridged segment was also simulated. For comparison, a monosegmental rigid fixation device as well as the effect of implant stiffness on intersegmental rotation were studied. The model was loaded with the upper body weight and muscle forces to simulate the four loading cases standing, 30° flexion, 20° extension, and 10° axial rotation. Intersegmental rotations, intradiscal pressure and facet joint forces were calculated at implant level and at the adjacent level above the implant. Implant forces were also determined. Compared to an intact spine, a dynamic implant reduces intersegmental rotation at implant level, decreases intradiscal pressure in a healthy disc for extension and standing, and decreases facet joint forces at implant level. With a rigid implant, these effects are more pronounced. With a slightly degenerated disc intersegmental rotation at implant level is mildly increased for extension and axial rotation and intradiscal pressure is strongly reduced for extension. After distraction, intradiscal pressure values are markedly reduced only for the rigid implant. At the adjacent level L2/L3, a posterior implant has only a minor effect on intradiscal pressure. However, it increases facet joint forces at this level for axial rotation and extension. Posterior implants are mostly loaded in compression. Forces in the implant are generally higher in a rigid fixator than in a dynamic implant. Distraction strongly increases both axial and shear forces in the implant. A stiffness of the implant greater than 1,000 N/mm has only a minor effect on intersegmental rotation. The mechanical effects of a dynamic implant are similar to those of a rigid fixation device, except after distraction, when intradiscal pressure is considerably lower for rigid than for dynamic implants. Thus, the results of this study demonstrate that a dynamic implant does not necessarily reduce axial spinal loads compared to an un-instrumented spine.
PMCID: PMC2200767  PMID: 17206401
Lumbar spine; Posterior dynamic implant; Internal fixation device; Finite element method; Biomechanics
7.  Comparison of BMP-2 and combined IGF-I/TGF-ß1 application in a sheep cervical spine fusion model 
European Spine Journal  2002;11(5):482-493.
Growth factors have proven to promote spine fusion. However, no comparative evaluation of growth factors in spinal fusion has yet been performed. The purpose of this study was to compare the efficacy and safety of combined IGF-I and TGF-ß1 application with BMP-2 application and autologous cancellous bone graft at an early time point in a sheep cervical spine fusion model. Thirty-two sheep underwent C3/4 discectomy and fusion. They were divided into four groups, according to their treatment: group 1, titanium cage (n=8); group 2, titanium cage filled with autologous cancellous iliac crest bone grafts (n=8); group 3, titanium cage coated with a poly-(D,L-lactide) (PDLLA) carrier including BMP-2 (5% w/w) (n=8); group 4, titanium cage coated with a PDLLA carrier including IGF-I (5% w/w) and TGF-ß1 (1% w/w) (n=8). Blood samples, body weight and temperature were analysed. Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8 and 12 weeks. At the same time points, disc space height and intervertebral angle were measured. After 12 weeks, the animals were killed and fusion sites were evaluated using functional radiographic views in flexion and extension. Quantitative computed tomographic scans were performed to assess bone mineral density, bone mineral content and bony callus volume. Biomechanical testing was carried out and the values for range of motion, and neutral and elastic zone were determined. Histomorphological and histomorphometrical analysis were performed and polychrome sequential labelling was used to determine the time frame of new bone formation. The results showed that, in comparison to the group treated with the cage alone (group 1), the cage plus BMP-2 group (group 3) and the cage plus IGF-I and TGF-ß1 group (group 4) demonstrated a significantly higher fusion rate in radiographic findings, a higher biomechanical stability, a more advanced interbody fusion in histomorphometrical analysis, and an accelerated interbody fusion on fluorochrome sequence labelling. In comparison to the bone graft group (group 2), the BMP-2 (group 3) and IGF-I/TGF-ß1 group (group 4) showed significantly less residual motion on functional radiographic evaluation, higher bone mineral density of the callus and higher biomechanical stability in extension, rotation and bending. The BMP-2 group showed significantly less residual motion on functional radiographic evaluation and higher intervertebral bone matrix formation on fluorochrome sequence labelling at 9 weeks in comparison to the IGF-I/TGF-ß1 group. In contrast, the IGF-I/TGF-ß1 group showed a significantly higher bone mineral density of the callus than the BMP-2 group. In comparison to the autologous cancellous bone graft group, both growth factors (BMP-2 and combined IGF-I and TGF-ß1) significantly improved the biomechanical results of interbody fusion. No systemic side effects were observed for either growth factor. On the basis of these preliminary results, it would appear that combined IGF-I/TGF-ß1 application yields equivalent results to BMP-2 application at an early time point in anterior sheep cervical spine fusion.
PMCID: PMC3611307  PMID: 12384758
Cervical spine Sheep Animal model Interbody fusion BMP-2 IGF-I TGF-ß1 Growth factor
8.  A new lumbar posterior fixation system, the memory metal spinal system: an in-vitro mechanical evaluation 
Spinal systems that are currently available for correction of spinal deformities or degeneration such as lumbar spondylolisthesis or degenerative disc disease use components manufactured from stainless steel or titanium and typically comprise two spinal rods with associated connection devices (for example: DePuy Spines Titanium Moss Miami Spinal System). The Memory Metal Spinal System of this study consists of a single square spinal rod made of a nickel titanium alloy (Nitinol) used in conjunction with connecting transverse bridges and pedicle screws made of Ti-alloy. Nitinol is best known for its shape memory effect, but is also characterized by its higher flexibility when compared to either stainless steel or titanium. A higher fusion rate with less degeneration of adjacent segments may result because of the elastic properties of the memory metal. In addition, the use of a single, unilateral rod may be of great value for a TLIF procedure. Our objective is to evaluate the mechanical properties of the new Memory Metal Spinal System compared to the Titanium Moss Miami Spinal System.
An in-vitro mechanical evaluation of the lumbar Memory Metal Spinal System was conducted. The test protocol followed ASTM Standard F1717-96, “Standard Test Methods for Static and Fatigue for Spinal Implant Constructs in a Corpectomy Model.”
1. Static axial testing in a load to failure mode in compression bending,
2. Static testing in a load to failure mode in torsion,
3. Cyclical testing to estimate the maximum run out load value at 5.0 x 10^6 cycles.
In the biomechanical testing for static axial compression bending there was no statistical difference between the 2% yield strength and the stiffness of the two types of spinal constructs.
In axial compression bending fatigue testing, the Memory Metal Spinal System construct showed a 50% increase in fatigue life compared to the Titanium Moss Miami Spinal System.
In static torsional testing the Memory Metal Spinal System constructs showed an average 220% increase in torsional yield strength, and an average 30% increase in torsional stiffness.
The in-vitro mechanical evaluation of the lumbar Memory Metal Spinal System showed good results when compared to a currently available spinal implant system. Throughout testing, the Memory Metal Spinal System showed no failures in static and dynamic fatigue.
PMCID: PMC3871762  PMID: 24047109
Memory metal spinal system; NiTi; DePuy spines titanium moss Miami; In-vitro mechanical evaluation; ASTM standard F1717-96
9.  Quantitative, Structural and Image-based Mechanical Analysis of Nonunion Fracture Repaired by Genetically Engineered Mesenchymal Stem Cells 
Journal of biomechanics  2010;43(12):2315-2320.
Stem cell-mediated gene therapy for fracture repair, utilizes genetically engineered mesenchymal stem cells (MSCs) for the induction of bone growth and is considered a promising approach in skeletal tissue regeneration. Previous studies have shown that murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post treatment. In the present study we sought to investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs. High resolution micro computed tomography (Micro-CT) was performed 10 and 35 weeks post MSC implantation, followed by micro finite element (Micro-FE) analysis. The results have shown that the regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume and connectivity density combined with an increase in mineral density. In addition, the axial stiffness of limbs repaired with MSCs was 2 to 1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post treatment. These results could be attributed to the fusion that occurred between in the ulna and radius bones. In conclusion, although MSCs induce bone formation, which exceeds the fracture site, significant remodeling of the repair callus occurs over time. In addition, limbs treated with an MSC graft demonstrated superior biomechanical properties, which could indicate the clinical benefit of future MSC application in nonunion fracture repair.
PMCID: PMC2948956  PMID: 20471652
Micro finite element model; Micro computed tomography; Bone tissue regeneration; Mesenchymal stem cells
10.  Benefits of biphasic calcium phosphate hybrid scaffold-driven osteogenic differentiation of mesenchymal stem cells through upregulated leptin receptor expression 
The use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation.
MSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT).
Tested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group.
The BCP hybrid scaffold for osteogenesis of MSCs is better than the coralline HA hybrid scaffold by upregulating expression of leptin-R. This was consistent with in vivo data, which indicated that BCP hybrid scaffolds induced more bone formation in a spinal fusion model.
PMCID: PMC4506435  PMID: 26179165
Coralline HA; BCP; MSCs; Osteogenesis; Leptin receptor
11.  Alendronate treatment improves bone–pedicle screw interface fixation in posterior lateral spine fusion: An experimental study in a porcine model 
International Orthopaedics  2009;34(3):447-451.
The bone–screw interface has been indicated as the weak link in pedicle screw spine fixation. Bisphosphonate treatment may have the effect of improving bone–screw interface fixation in spine fusion by inhibiting bone resorption. An experimental study was conducted using a porcine model to evaluate the influence of alendronate treatment on bone–pedicle screw interface fixation. Eleven pigs in the treatment group received alendronate 10 mg/day orally for three months postoperatively. The other 11 pigs served as a control group. Posterior lateral fusion with the CD Horizon pedicle screw system was performed with autograft on the lumbar spine on all animals. Biomechanical torsion test and histomorphometric parameters of screw fixation were evaluated three months after the operation. The maximum torque and initial angular stiffness of the treatment group was higher than that of the control group, but there was no statistical significance. The bone–screw contact surface was 23.3 ± 10% for the treatment group and 9.8 ± 5.9% for the control group (P < 0.01). This study indicated that alendronate treatment increased bone purchase of stainless steel screw surfaces.
PMCID: PMC2899306  PMID: 19330331
12.  Efficacy of interspinous process lumbar fusion with recombinant human bone morphogenetic protein-2 delivered with a synthetic polymer and β-tricalcium phosphate in a rabbit model 
European Spine Journal  2011;21(7):1338-1345.
As a powerful bone-inducing cytokine, rhBMP-2 has been used as a bone graft substitute in combination with animal-derived collagen to achieve interbody or posterolateral spinal fusion. Successful interspinous process fusion using rhBMP-2 in combination with synthetic carrier materials would offer a safe, minimally invasive spinal fusion option for the treatment of spinal disorders. The aims of the present study were to achieve interspinous process fusion by implanting rhBMP-2-retaining degradable material instead of bone grafting and to evaluate efficacy for vertebral stabilization.
Materials and methods
A polymer gel (200 mg), β-tricalcium phosphate powder (400 mg), and rhBMP-2 (0, 30, 60 or 120 μg) were mixed to generate a plastic implant, which was then placed during surgery to bridge the L5–6 interspinous processes of 58 rabbits. Control animals received implants either without rhBMP-2 or with autogenous bone chips from the iliac crest. L5–6 vertebrae were recovered 8 weeks postoperatively. Interspinous process fusion was evaluated by radiography, biomechanical bending test, intradiscal pressure (IDP) measurement, and histology.
In bending tests, strength of fusion was significantly greater in BMP60 and BMP120 groups than in sham, BMP0, BMP30 or autogenous bone groups. IDP at L5–6 was significantly reduced in BMP60 and BMP120 groups compared to sham, BMP0, BMP30, and autograft groups. Histologically, coronal sections of the fusion mass showed a bone mass bridging both spinous processes.
Solid interspinous process fusion was achieved in rabbit models by 8 weeks after implanting the biodegradable bone-inducing material. These results suggest a potential new less-invasive option without bone grafting for the treatment of lumbar disorders.
PMCID: PMC3389107  PMID: 22203476
Animal model; Interspinous process spine fusion; Minimally invasive surgery; Recombinant human bone morphogenetic protein-2
13.  Reprogramming of mesenchymal stem cells derived from iPSCs seeded on biofunctionalized calcium phosphate scaffold for bone engineering 
Biomaterials  2013;34(32):10.1016/j.biomaterials.2013.07.029.
Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a promising choice of patient-specific stem cells with superior capability of cell expansion. There has been no report on bone morphogenic protein 2 (BMP2) gene modification of iPSC-MSCs for bone tissue engineering. The objectives of this study were to: (1) genetically modify iPSC-MSCs for BMP2 delivery; and (2) to seed BMP2 gene-modified iPSC-MSCs on calcium phosphate cement (CPC) immobilized with RGD for bone tissue engineering. iPSC-MSCs were infected with green fluorescence protein (GFP-iPSC-MSCs), or BMP2 lentivirus (BMP2-iPSC-MSCs). High levels of GFP expression were detected and more than 68% of GFP-iPSC-MSCs were GFP positive. BMP2-iPSC-MSCs expressed higher BMP2 levels than iPSC-MSCs in quantitative RT-PCR and ELISA assays (p < 0.05). BMP2-iPSC-MSCs did not compromise growth kinetics and cell cycle stages compared to iPSC-MSCs. After 14 d in osteogenic medium, ALP activity of BMP2-iPSC-MSCs was 1.8 times that of iPSC-MSCs (p < 0.05), indicating that BMP2 gene transduction of iPSC-MSCs enhanced osteogenic differentiation. BMP2-iPSC-MSCs were seeded on CPC scaffold biofunctionalized with RGD (RGD-CPC). BMP2-iPSC-MSCs attached well on RGD-CPC. At 14 d, COL1A1 expression of BMP2-iPSC-MSCs was 1.9 times that of iPSC-MSCs. OC expression of BMP2-iPSC-MSCs was 2.3 times that of iPSC-MSCs. Bone matrix mineralization by BMP2-iPSC-MSCs was was 1.8 times that of iPSC-MSCs at 21 d. In conclusion, iPSC-MSCs seeded on CPC were suitable for bone tissue engineering. BMP2 gene-modified iPSC-MSCs on RGD-CPC underwent osteogenic differentiation, and the overexpression of BMP2 in iPSC-MSCs enhanced differentiation and bone mineral production on RGD-CPC. BMP2-iPSC-MSC seeding on RGD-CPC scaffold is promising to enhance bone regeneration efficacy.
PMCID: PMC3845377  PMID: 23891395
bone morphogenetic protein 2 (BMP2); bone tissue engineering; calcium phosphate cement (CPC); gene transduction; induced pluripotent stem cells (iPSCs); RGD immobilization
14.  The Use of Bone Morphogenetic Protein in Pediatric Cervical Spine Fusion Surgery: Case Reports and Review of the Literature 
Global Spine Journal  2015;6(1):e41-e46.
Study Design Case report.
Objective There is a paucity of literature describing the use of bone graft substitutes to achieve fusion in the pediatric cervical spine. The outcomes and complications involving the off-label use of bone morphogenetic protein (BMP)-2 in the pediatric cervical spine are not clearly defined. The purpose of this article is to report successful fusion without complications in two pediatric patients who had instrumented occipitocervical fusion using low-dose BMP-2.
Methods A retrospective review of the medical records was performed, and the patients were followed for 5 years. Two patients under 10 years of age with upper cervical instability were treated with occipitocervical instrumented fusion using rigid occipitocervical fixation techniques along with conventionally available low-dose BMP-2. A Medline and PubMed literature search was conducted using the terms “bone morphogenetic protein,” “BMP,” “rh-BMP2,” “bone graft substitutes,” and “pediatric cervical spine.”
Results Solid occipitocervical fusion was achieved in both pediatric patients. There were no reported perioperative or follow-up complications. At 5-year follow-up, radiographs in both patients showed successful occipital cervical fusion without evidence of instrumentation failure or changes in the occipitocervical alignment. To date, there are few published reports on this topic. Complications and the appropriate dosage application in the pediatric posterior cervical spine remain unknown.
Conclusions We describe two pediatric patients with upper cervical instability who achieved successful occipital cervical fusion without complication using off-label BMP-2. This report underscores the potential for BMP-2 to achieve successful arthrodesis of the posterior occipitocervical junction in pediatric patients. Use should be judicious as complications and long-term outcomes of pediatric BMP-2 use remain undefined in the existing literature.
PMCID: PMC4733381  PMID: 26835215
BMP; bone morphogenic protein; rh-BMP-2; bone graft substitutes; pediatric cervical spine
15.  Posterolateral inter-transverse lumbar fusion in a mouse model 
Spinal fusion is a common orthopaedic procedure that has been previously modeled using canine, lapine, and rodent subjects. Despite the increasing availability of genetically modified mouse strains, murine models have only been infrequently described.
To present an efficient and minimally traumatic procedure for achieving spinal fusion in a mouse model and determine the optimal rhBMP-2 dose to achieve sufficient fusion mass.
MicroCT reconstructions of the unfused mouse spine and human spine were compared to design a surgical approach. In phase 1, posterolateral lumbar spine fusion in the mouse was evaluated using 18 animals allocated to three experimental groups. Group 1 received decortication only (n = 3), Group 2 received 10 μg rhBMP-2 in a collagen sponge bilaterally (n = 6), and Group 3 received 10 μg rhBMP-2 + decortication (n = 9). The surgical technique was assessed for intra-operative safety, efficacy, access and reproducibility. Spines were harvested for analysis at 3 weeks (Groups 1, 2) and 1, 2, and 3 weeks (Group 3). In phase 2, a dose response study was carried out in an additional 18 animals with C57BL6 mice receiving sponges containing 0, 0.5, 1, 2.5, 5 μg of rhBMP-2 per sponge bilaterally.
The operative procedure via midline access was rapid and reproducible, and fusion of the murine articular processes was found to be analogous to the human procedure. Unlike reports from other species, decortication alone (Group 1) yielded no new bone formation. Addition of rhBMP-2 (Groups 2 and 3) yielded a significant bone mass that bridged the L4-L6 vertebrae. The subsequent dose response experiment revealed that 0.5 μg rhBMP-2 per sponge was sufficient to create a fusion mass.
We describe a new approach for mouse lumbar spine fusion that is safe, efficient, and highly reproducible. The technique we employed is analogous to the human midline procedure and may be highly suitable for genetically modified mouse models.
PMCID: PMC3564784  PMID: 23342962
Spine; Fusion; Arthrodesis; Mouse; BMP
16.  Biomechanical evaluation of the Total Facet Arthroplasty System® (TFAS®): loading as compared to a rigid posterior instrumentation system 
European Spine Journal  2012;21(8):1660-1673.
To gain insight into a new technology, a novel facet arthroplasty device (TFAS) was compared to a rigid posterior fixation system (UCR). The axial and bending loads through the implants and at the bone-implant interfaces were evaluated using an ex vivo biomechanical study and matched finite element analysis. Kinematic behaviour has been reported for TFAS, but implant loads have not. Implant loads are important indicators of an implant’s performance and safety. The rigid posterior fixation system is used for comparison due to the extensive information available about these systems.
Unconstrained pure moments were applied to 13 L3–S1 cadaveric spine segments. Specimens were tested intact, following decompression, UCR fixation and TFAS implantation at L4–L5. UCR fixation was via standard pedicle screws and TFAS implantation was via PMMA-cemented transpedicular stems. Three-dimensional 10 Nm moments and a 600 N follower load were applied; L4–L5 disc pressures and implant loads were measured using a pressure sensor and strain gauges, respectively. A finite element model was used to calculate TFAS bone-implant interface loads.
UCR experienced greater implant loads in extension (p < 0.004) and lateral bending (p < 0.02). Under flexion, TFAS was subject to greater implant moments (p < 0.04). At the bone-implant interface, flexion resulted in the smallest TFAS (average = 0.20 Nm) but greatest UCR (1.18 Nm) moment and axial rotation resulted in the greatest TFAS (3.10 Nm) and smallest UCR (0.40 Nm) moments. Disc pressures were similar to intact for TFAS but not for UCR (p < 0.04).
These results are most applicable to the immediate post-operative period prior to remodelling of the bone-implant interface since the UCR and TFAS implants are intended for different service lives (UCR—until fusion, TFAS—indefinitely). TFAS reproduced intact-like anterior column load-sharing—as measured by disc pressure. The highest bone-implant moment of 3.1 Nm was measured in TFAS and for the same loading condition the UCR interface moment was considerably lower (0.4 Nm). For other loading conditions, the differences between TFAS and UCR were smaller, with the UCR sometimes having larger values and for others the TFAS was larger. The long-term physiological meaning of these findings is unknown and demonstrates the need for a better understanding of the relationship between spinal arthroplasty devices and the host tissue as development of next generation motion-preserving posterior devices that hope to more accurately replicate the natural functions of the native tissue continues.
PMCID: PMC3535260  PMID: 22407270
Total facet arthroplasty; Implant loading; Bone-implant interface; Load sharing; Lumbar spine
17.  In vitro testing of a new transpedicular stabilization technique 
European Spine Journal  1997;6(4):249-255.
The rigidity of a pedicle screw implant is a critical biomechanical variable in lumbar spinal fusions. Sufficient rigidity is required for integration of bone grafts and to promote healing. Osteopenia, stress shielding, and compensatory hypermobility have been described as consequences of excessive rigidity. Little is known about the biomechanical characteristics of “semirigid” compared to “rigid” implants. A new implant, whose rigidity can be varied by selection of different implant components, was tested in vitro under well-defined loading conditions. The three-dimensional load-displacement behavior of all lumbar vertebrae involved in or adjacent to the two-level fusion was evaluated for two fusion modifications: bilateral rigid and bilateral semirigid. Cyclic fatigue loading was subsequently carried out under realistic conditions and motion testing repeated. The rigid device reduced the motion of the L3–4 transfixed segment in the primary movement planes by 87.3% with respect to the intact spine value in flexion/extension (FE), 86.3% in lateral bending (LB), and 76.8% in axial rotation (AR). The semirigid device achieved a reduction in motion of 79.6% (FE), 82.7% (LB), and 51.7% (AR). The semirigid implant was particularly easy to insert, because no bending of rods or plates was necessary. The implants showed no loosening or breakage after the fatigue testing. The results are compared to other available systems and the underlying biomechanics discussed.
PMCID: PMC3454640  PMID: 9294749
Spine; Spinal fusion; Biomechanics; Pedicle screw; Stress shielding
18.  Anatomic Facet Replacement System (AFRS) Restoration of Lumbar Segment Mechanics to Intact: A Finite Element Study and In Vitro Cadaver Investigation 
SAS Journal  2007;1(1):46-54.
Many decompression procedures involve complete or partial facetectomy. Spinal fusion usually stabilizes the motion segment after complete facetectomies. However, problems with fusion, such as adjacent-level degeneration, have increased interest in motion- preservation technologies. Facet arthroplasty may become an important posterior motion-preservation device, but its biomechanical literature is sparse.
We conducted an in vitro investigation and finite element study to compare the biomechanical effects of an artificial facet system to the intact spine. In the in vitro study, we tested human osteo-ligamentous segments (L3-S1) in intact, injured, and artificial facet–repaired conditions. For the finite element study, we used a 3-dimensional ligamentous L3-S1 segment model. We simulated destabilization in the intact model by removing the facets across the L4-L5 functional unit, then repaired it with appropriately sized facet implants and compared the ranges of motion, facet loads, disc pressures, and device loads. We also analyzed a finite element model with a rigid posterior pedicle-rod fixation system. We subjected the cadaveric specimens and the models to 400 N of follower load plus a 10 Nm moment in extension, flexion, bending, and rotation. We used a novel technique to apply the follower load in the finite element models such that preload induced minimal vertebral rotation during the range of motion.
The predicted ranges of motion for the intact and implanted models were consistent with cadaver data. After destabilization and facet replacement, the artificial facet system restored motion in all loading modes to intact values. The implant facet loads were similar to intact facet loads in extension and axial rotation, but less in lateral bending. The intradiscal pressure at the implanted level for the facet replacement device was similar to the intact pressure, whereas with the rigid system the intradiscal pressure was up to 70% less than the intact pressure. The maximum von-Mises stress predicted in the facet replacement construct was 85 MPa in extension at the bone–pedicle screw interface, compared with 174 MPa in the rigid system. Contact stresses at implant mating surfaces were minimal.
The artificial facet system replicated natural facet kinematics. The cadaveric ranges of motion and the predicted finite element–based data indicated that the implant can “restore” the normal function of the segment after artificial facet replacement.
Clinical Relevance
Compared to rigid posterior pedicle-rod fixation, the artificial facet system restored the intact mechanics at the implanted level and may prevent adjacent-level degeneration.
PMCID: PMC4365566  PMID: 25802578
artificial facet; lumbar spine; biomechanics; finite element technique
19.  Fusion and failure following anterior cervical plating with dynamic or rigid plates: 6-months results of a multi-centric, prospective, randomized, controlled study 
European Spine Journal  2007;16(10):1689-1694.
Anterior cervical plate fixation is an approved surgical technique for cervical spine stabilization in the presence of anterior cervical instability. Rigid plate design with screws rigidly locked to the plate is widely used and is thought to provide a better fixation for the treated spinal segment than a dynamic design in which the screws may slide when the graft is settling. Recent biomechanical studies showed that dynamic anterior plates provide a better graft loading possibly leading to accelerated spinal fusion with a lower incidence of implant complications. This, however, was investigated in vitro and does not necessarily mean to be the case in vivo, as well. Thus, the two major aspects of this study were to compare the speed of bone fusion and the rate of implant complications using either rigid- or dynamic plates. The study design is prospective, randomized, controlled, and multi-centric, having been approved by respective ethic committees of all participating sites. One hundred and thirty-two patients were included in this study and randomly assigned to one of the two groups, both undergoing routine level-1- or level-2 anterior cervical discectomy with autograft fusion receiving either a dynamic plate with screws being locked in ap - position (ABC, Aesculap, Germany), or a rigid plate (CSLP, Synthes, Switzerland). Segmental mobility and implant complications were compared after 3- and 6 months, respectively. All measurements were performed by an independent radiologist. Mobility results after 6 months were available for 77 patients (43 ABC/34 CSLP). Mean segmental mobility for the ABC group was 1.7 mm at the time of discharge, 1.4 mm after 3 months, and 0.8 mm after 6 months. For the CSLP- group the measurements were 1.0, 1.8, and 1.7 mm, respectively. The differences of mean segmental mobility were statistically significant between both groups after 6 months (P = 0.02). Four patients of the CSLP-group demonstrated surgical hardware complications, whereas no implant complications were observed within the ABC-group (P = 0.0375). Dynamic plate designs provided a faster fusion of the cervical spine compared with rigid plate designs after prior spinal surgery. Moreover, the rate of implant complications was lower within the group of patients receiving a dynamic plate. These interim results refer to a follow-up period of 6 months after prior spinal surgery. Further investigations will be performed 2 years postoperatively.
PMCID: PMC2078301  PMID: 17684777
Cervical Spine; Anterior plates; Implant; Randomized controlled study
20.  Evaluation of Posterolateral Lumbar Fusion in Sheep Using Mineral Scaffolds Seeded with Cultured Bone Marrow Cells 
The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft) for posterolateral lumbar fusion (PLF) in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM) mesenchymal stem cells (MSCs) have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group), hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct). During the last three days of culture, dexamethasone (dex) and beta-glycerophosphate (β-GP) were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4–L5). Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT), histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70%) than for mineral scaffold alone (22%) and hybrid constructs (35%). The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft). Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored by bioluminescence imaging (BLI). Although the cultured MSCs had osteogenic potential, their contribution to spinal fusion when seeded in mineral scaffolds, in the conditions disclosed here, remains uncertain probably due to callus interference with the scaffolds. At present, bone autografts are better than hybrid constructs for posterolateral lumbar fusion, but we should continue to seek better conditions for efficient tissue engineering.
PMCID: PMC4284771  PMID: 25522168
spinal fusion; autograft; allograft; mesenchymal stem cell; scaffold; hydroxyapatite; tissue engineering; callus; CT scan; histology; histomorphometry
21.  Human Perivascular Stem Cell-Based Bone Graft Substitute Induces Rat Spinal Fusion 
Stem Cells Translational Medicine  2014;3(10):1231-1241.
This study evaluated the osteogenic potential of freshly sorted human perivascular stem cells (hPSCs) without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography, suggesting that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair.
Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair.
PMCID: PMC4181396  PMID: 25154782
Perivascular stem cell; Adventitial cell; Mesenchymal stem cell; Osteogenesis; Pericyte; Tissue engineering
22.  Enhanced Control of In Vivo Bone Formation with Surface Functionalized Alginate Microbeads Incorporating Heparin and Human Bone Morphogenetic Protein-2 
Tissue Engineering. Part A  2012;19(3-4):350-359.
In this study, we tested the hypothesis that a surface functionalization delivery platform incorporating heparin onto strontium alginate microbeads surfaces would convert this “naive carriers” into “mini-reservoirs” for localized in vivo delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) that will induce functional bone regeneration. In vitro evaluation confirmed that (1) heparin incorporation could immobilize and prolong rhBMP-2 release for approximately 3 weeks; (2) a significant decrease (p<0.01) in rhBMP-2 burst release is attainable depending on initial protein load; and (3) rhBMP-2 released from surface functionalized microbeads retained bioactivity and stimulated higher alkaline phosphatase activity in cultured C2C12 cells when compared with daily administration of fresh bolus rhBMP-2. Subsequently, surface functionalized microbeads were used for in vivo delivery of rhBMP-2 at local sites of posterolateral spinal fusion surgery in rats. The microbeads were loaded into the pores of medical-grade polyepsilone caprolactone-tricalcium phosphate scaffolds before implantation. Results revealed robust bone formation and a biomechanically solid fusion after 6 weeks. When compared with a control group consisting of an equivalent amount of rhBMP-2 that was directly adsorbed onto bare-surfaced microbeads with no heparin, a 5.3-fold increase in bone volume fraction and a 2.6-fold increase in bending stiffness (flexion/extension) were observed. When compared with collagen sponge carriers of rhBMP-2, a 1.5-fold and a 1.3-fold increase in bone volume fraction and bending stiffness were observed, respectively. More importantly, 3D micro-computed tomography images enabled the visualization of a well-contained newly formed bone at ipsilateral implant sites with surface functionalized rhBMP-2 delivery. This was absent with collagen sponge carriers where newly formed bone tissue was poorly contained and crossed over the posterior midline to contralateral implants. These findings are important because of complications with current rhBMP-2 delivery method, including excessive, uncontrolled bone formation.
PMCID: PMC3542875  PMID: 22894570
23.  Is a single anterolateral screw-plate fixation sufficient for the treatment of spinal fractures in the thoracolumbar junction? A Biomechanical in vitro Investigation 
European Spine Journal  2004;14(2):197-204.
Controversy exists about the indications, advantages and disadvantages of various surgical techniques used for anterior interbody fusion of spinal fractures in the thoracolumbar junction. The purpose of this study was to evaluate the stabilizing effect of an anterolateral and thoracoscopically implantable screw-plate system. Six human bisegmental spinal units (T12–L2) were used for the biomechanical in vitro testing procedure. Each specimen was tested in three different scenarios: (1) intact spinal segments vs (2) monosegmental (T12/L1) anterolateral fixation (macsTL, Aesculap, Germany) with an interbody bone strut graft from the iliac crest after both partial corpectomy (L1) and discectomy (T12/L1) vs (3) bisegmental anterolateral instrumentation after extended partial corpectomy (L1), and bisegmental discectomy (T12/L1 and L1/L2). Specimens were loaded with an alternating, nondestructive maximum bending moment of ±7.5 Nm in six directions: flexion/extension, right and left lateral bending, and right and left axial rotation. Motion analysis was performed by a contact-less three-dimensional optical measuring system. Segmental stiffness of the three different scenarios was evaluated by the relative alteration of the intervertebral angles in the three main anatomical planes. With each stabilization technique, the specimens were more rigid, compared with the intact spine, for flexion/extension (sagittal plane) as well as in left and right lateral bending (frontal plane). In these planes the bisegmental instrumentation compared to the monosegmental case had an even larger stiffening effect on the specimens. In contrast to these findings, axial rotation showed a modest increase of motion after bisegmental instrumentation. To conclude, the immobilization of monosegmental fractures in the thoracolumbar junction can be secured by means of bone grafting and the implant used in this study for all three anatomical planes. After bisegmental anterolateral stabilization a sufficient reduction of the movements was registered for flexion/extension and lateral bending. However, the observed slight increase of the range of motion in the transversal plane may lead to loosening of the implant before union. Therefore, the use of an additional dorsal fixation device should be considered.
PMCID: PMC3476694  PMID: 15243790
Spine; Biomechanics; In vitro testing
24.  Comparison of a Novel Oxysterol Molecule and rhBMP2 Fusion Rates in a Rabbit Posterolateral Lumbar Spine Model 
Background Context
The non-union rate following lumbar spinal fusion is as high as 25%. Bone morphogenetic protein-2 (rhBMP2) has been used as a biological adjunct to promote bony fusion. However, recently there have been concerns about BMP2. Oxysterol 133 (Oxy133) has been shown to promote excellent fusion rates in rodent lumbar spine models and offers a potential alternative to rhBMP2.
The purpose of this study was to compare the fusion rate of rhBMP2 and Oxy133 in a randomized controlled trial using a posterolateral lumbar rabbit spinal fusion model.
Study Design
This was a randomized control animal study.
Twenty-four male adult white New Zealand rabbits (3–3.5kg) underwent bilateral posterolateral lumbar spinal fusion at L4–L5. Rabbits were divided into 4 groups: control (A), 30 µg rhBMP2 (B), 20 mg Oxy133 (C), and 60 mg Oxy133 (D). At 4 weeks, fusion was evaluated by fluoroscopy, and at 8 weeks the rabbits were sacrificed and fusion was evaluated radiographically, by manual palpation, and with microCT. Dr. Parhami is a founder and Dr. Stappenbeck is the Director of Chemistry at MAX BioPharma, which has licensed the rights to Oxy133 from UCLA, both have financial interests in the technology presented here. UCLA holds equity in MAX BioPharma. All other authors have no conflicts of interest. Studies reported here were supported in part by the NIH/NIAMS grant RO1AR059794 and in part by MAX BioPharma that purchased the rabbits and provided Oxy133.
Fusion rates by radiographic analysis at 8 weeks were: group A 40.0%, group B 91.7%, group C 91.7%, and group D 100%. Evaluation of fusion masses by manual palpation of excised spines after sacrifice showed the following fusion rates: group A 0%, group B 83.3%, group C 83.3%, and group D 90%. MicroCT scanning confirmed these findings.
These findings in a rabbit model demonstrate that both 20 mg dose and 60 mg dose Oxy133 promote fusion that is equivalent to fusion induced by 30 µg rhBMP2 and significantly greater than the control group. The present findings confirm that Oxy133 is a promising candidate for therapeutic development as an alternative to rhBMP2 to promote spinal fusion.
PMCID: PMC4392926  PMID: 25450659
25.  Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells 
Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family.
Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry.
Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs.
We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.
PMCID: PMC4727301  PMID: 26809343
Spinal non-fusion; Osteogenesis; Bone marrow; Mesenchymal stem cells; Human nucleus pulposus cells; Human annulus fibrosus cells; Relative gene expression

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