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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.  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
6.  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
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.  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
9.  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
10.  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
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.  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
14.  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
15.  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
16.  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
17.  Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction 
World Journal of Stem Cells  2015;7(1):223-234.
AIM: To investigate adenoviral transduction in mesenchymal stem cells (MSCs) and effects on stemness in vitro and function as a cell therapy in vivo.
METHODS: Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine (PEI)-mediated transfection of pcDNA3-eGFP or adenoviral transduction of green fluorescent protein (GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness (i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2 (BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology.
RESULTS: PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs (81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs (78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs (7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression (0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs (1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitro were not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult (P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells (P < 0.05). Ad-BMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs (83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone (1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05).
CONCLUSION: Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.
PMCID: PMC4300933  PMID: 25621122
Adenovirus; Bone morphogenic protein 2; Fetal; Green fluorescent protein; Mesenchymal stem cell; Gene transfer technique
18.  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
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.  BMP-13 Emerges as a Potential Inhibitor of Bone Formation 
Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.
PMCID: PMC2646266  PMID: 19240811
BMP-13; GDF6; CDMP-2; osteogenic differentiation; mesenchymal stromal cells
21.  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
22.  Characterization and spinal fusion effect of rabbit mesenchymal stem cells 
BMC Research Notes  2013;6:528.
The surface markers of mesenchymal stem cells (MSCs) of rabbits have been reported only sporadically. However, interest in the spinal fusion effect of MSCs has risen recently. The purpose of this research was to study the surface markers and spinal fusion effect of rabbit MSCs.
Of our rabbit MSCs, 2% expressed CD14, CD29, and CD45, 1% expressed CD90 and 97% expressed CD44. These results implied the MSCs were negative for CD14, CD29, CD45, and CD90, but positive for CD44. The surgical results showed that satisfactory fusion occurred in 10 rabbits (83%) in the study group and unsatisfactory fusion in 2 (17%). In the control group, satisfactory fusion was found in 3 rabbits (25%) and unsatisfactory fusion in 9 (75%). Statistical analysis showed the study group had significantly better spinal fusion results than the control group.
The surface markers of human and rabbit MSCs are not exactly the same. Rabbit MSCs do not have positive reactivity for CD29 and CD90, which are invariably present on human MSCs. The allogeneic undifferentiated rabbit MSCs were able to promote spinal fusion and did not induce an adverse immune response.
PMCID: PMC4029367  PMID: 24325928
Characterization; Spinal fusion; Mesenchymal stem cell
23.  BMP-Non-Responsive Sca1+CD73+CD44+ Mouse Bone Marrow Derived Osteoprogenitor Cells Respond to Combination of VEGF and BMP-6 to Display Enhanced Osteoblastic Differentiation and Ectopic Bone Formation 
PLoS ONE  2014;9(7):e103060.
Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.
PMCID: PMC4105618  PMID: 25048464
24.  Posterior lumbar interbody fusion using one diagonal fusion cage with transpedicular screw/rod fixation 
European Spine Journal  2003;12(2):173-177.
Posterior lumbar interbody fusion (PLIF) using threaded cages has gained wide popularity for lumbosacral spinal disease. Our biomechanical tests showed that PLIF using a single diagonal cage with unilateral facetectomy does add a little to spinal stability and provides equal or even higher postoperative stability than PLIF using two posterior cages with bilateral facetectomy. Studies also demonstrated that cages placed using a posterior approach did not cause the same increase in spinal stiffness seen with pedicle screw instrumentation, and we concluded that cages should not be used posteriorly without other forms of fixation. On the other hand, placement of two cages using a posterior approach does have the disadvantage of risk to the bilateral nerve roots. We therefore performed a prospective study to determine whether PLIF can be accomplished by utilizing a single diagonal fusion cage with the application of supplemental transpedicular screw/rod instrumentation. Twenty-seven patients underwent a PLIF using one single fusion cage (BAK, Sulzer Spine-Tech, Minneapolis, MN, USA) inserted posterolaterally and oriented anteromedially on the symptomatic side with unilateral facetectomy and at the same level supplemental fixation with a transpedicular screw/rod system. The internal fixation systems included 12 SOCON spinal systems (Aesculap AG, Germany) and 15 TSRH spinal systems (Medtronic Sofamor Danek, USA). The inclusion criteria were grade 1 to 2 lumbar isthmic spondylolisthesis, lumbar degenerative spondylolisthesis, and recurrent lumbar disc herniations with instability. Patients had at least 1 year of low back pain and/or unilateral sciatica and a severely restricted functional ability in individuals aged 28–55 years. Patients with more than grade 2 spondylolisthesis or adjacent-level degeneration were excluded from the study. Patients were clinically assessed prior to surgery by an independent assessor; they were then reassessed at 1, 3, 6, 12, 18, and 24 months postoperatively by the same assessor and put into four categories: excellent, good, fair, and poor. Operative time, blood loss, hospital expense, and complications were also recorded. All patients achieved successful radiographic fusion at 2 years, and this was achieved at 1 year in 25 out of 27 patients. At 2 years, clinical results were excellent in 15patients, good in 10, fair in 1, and poor in 1. Regarding complications, one patient had a postoperative motor and sensory deficit of the nerve root. Reoperation was required in one patient due to migration of pedicle screws. No implant fractures or deformities occurred in any of the patients. PLIF using diagonal insertion of a single threaded cage with supplemental transpedicular screw/rod instrumentation enables sufficient decompression and solid interbody fusion to be achieved with minimal invasion of the posterior spinal elements. It is a clinically safer, easier, and more economical means of accomplishing PLIF.
PMCID: PMC3784842  PMID: 12709855
Lumbar Fusion cage Implant Transpedicular screw Interbody
25.  Titanium-alloy enhances bone-pedicle screw fixation: mechanical and histomorphometrical results of titanium-alloy versus stainless steel 
European Spine Journal  2000;9(2):97-103.
Several types of pedicle screw systems have been utilized to augment lumbar spine fusion. The majority of these systems are made of stainless steel (Ss), but titanium-alloy (Ti-alloy) devices have recently been available on the market. Ti-alloy implants have several potential advantages over Ss ones. High bioactivity and more flexibility may improve bone ingrowth and mechanical fixation, and the material also offers superior magnetic resonance imaging (MRI) and computed tomography (CT) resolution and significantly less signal interference. However, no data are available from loaded spinal constructs regarding bony ingrowth and mechanical fixation. The aim of this study was to analyse the effect of Ti-alloy versus Ss pedicle screws on mechanical fixation and bone ingrowth in a loaded mini-pig model. Eighteen adult mini-pigs underwent total laminectomy and posterolateral spinal fusion at L3-L4, and were randomly selected to receive either Ss (n = 9) or Ti (n = 9) pedicle screw devices. In both groups, the device used was compact Cotrel-Dubousset instrumentation (Sofamore Danek) of an identical size and shape. The postoperative observation time was ¶3 months. Screws from L3 were used for histomorphometric studies. Mechanical testing (torsional tests and pull-out tests) was performed on the screws from L4. The Ti screws had a higher maximum torque (P < 0.05) and angular stiffness (P < 0.07), measured by torsional testing. In the pull-out tests, no differences were found between the two groups with respect to the maximum load, stiffness and energy to failure. No correlation between removal torque and the pull-out strength was found (r = 0.1). Bone ongrowth on Ti was increased by 33% compared with Ss ¶(P < 0.04), whereas no differences in bone volume around the screws were shown. Mechanical binding at the bone-screw interface was significantly greater for Ti pedicle screws than for Ss, which was explained by the fact that Ti screws had a superior bone ongrowth. There was no correlation between the screw removal torque and the pull-out strength, which indicates that the peripheral bone structure around the screw was unaffected by the choice of metal.
PMCID: PMC3611362  PMID: 10823424
Key words Pedicle screw; Titanium; Stainless steel; Pull-out test; Torsion; Histomorphometry

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